Alpha- and gamma-truxillic acid derivatives and pharmaceutical compositions thereof

ABSTRACT

The present invention provides a compound, and method of inhibiting the activity of a Fatty Acid Binding Protein (FABP) comprising contacting the FABP with a compound, having the structure:

The invention was made with government support under Grant numberDA032232, DA026953, and DA016419 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

Throughout this application, certain publications are referenced inparentheses. Full citations for these publications may be foundimmediately preceding the claims. The disclosures of these publicationsin their entireties are hereby incorporated by reference into thisapplication in order to describe more fully the state of the art towhich this invention relates.

BACKGROUND OF THE INVENTION

Lipids, owing to their water insolubility, require a variety of fattyacid binding protein (FABP) chaperones or transporters to carry themthroughout cells (Furuhashi, M et al. 2008). The FABPs are part of thepathway of anandamide inactivation by the fatty acid amide hydrolase(FAAH), an enzyme localized inside the cell on the endoplasmicreticulum. The endocannabinoid anandamide (arachidonoyl ethanolamide orAEA) is an uncharged neuromodulatory lipid that is inactivated throughits cellular uptake by FABPs and subsequent hydrolysis by FAAH intoethanolamine and arachidonic acid.

Cannabinoids such as anandamide have broad effects on the centralnervous system (CNS) and influence, for example, movement, memory,nociception, endocrine regulation, thermoregulation, sensory perception,cognitive functions, and mood. Similarly, genetic and pharmacologicalstudies have revealed a broad role for endocannabinoid signaling in avariety of physiological processes, including neuromodulator release,motor learning, synaptic plasticity, appetite, and pain sensation.Anandamide produces most of its pharmacological effects by binding andactivating the cannabinoid receptor (CB-1 and CB-2) within the CNS. Theincrease in extracellular anandamide caused by the inhibition of FABPstriggers activation of the cannabinoid receptor type 1 (CB-1) pathwayleading to the relief of neurogenic and inflammatory pain.

Recently, it was shown that anandamide (an endocannabinoid) uses FABPssuch as FABP5 (E-FABP) and FABP7 (B-FABP) as intracellular transporters(Kaczocha, M. et al. 2009). FABPs are drug targets similar to FAAH sinceinhibitors of each decrease hydrolysis of anandamide and its uptake intocells, raising the levels of extracellular anandamide (FIG. 1) (Howlett,A. C. et al. 2011; Kaczocha, M. et al. 2012; Ahn, K. et al. 2009). Fewspecific FABP inhibitors have been described. There are those that werespecifically designed for FABP4, such as BMS309403, which are importantfor the protective effects that they exert in metabolic syndrome andatherosclerosis (Barf, T. et al. 2009; Sulsky, R. et al. 2007).BMS309403 also binds other FABPs, such as FABP5 and FABP7, that carryanandamide, as do other inhibitors originally designed to inhibit aputative anandamide transmembrane transporter (Kaczocha, M. et al.2012).

SUMMARY OF THE INVENTION

The present invention provides a compound having the structure:

whereinR₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅,        —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NR₁₅R₁₆, -alykl-OR₁₅, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or        heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl,        cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl;        when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₇, R₈, R₉,        R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other        than —C(═O)OR₁₃ where R₁₃ is —CH₃, —CH₂CH₃, tolyl or propyl        1-bromo-1-methylpropanoyloxybutyl ester, or —C(═O)NR₁₃R₁₄ where        one of R₁₃ or R₁₄ is phenyl and the other is —H, or both of R₁₃        and R₁₄ are —H; and        when one of R₁ or R₂ is —C(═O) OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈,        R₉, R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is        other than —C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the        other is (2-methylmercapto)phenyl;        or an enantiomer or pharmaceutically acceptable salt thereof.

The present invention provides a method of inhibiting the activity of aFatty Acid Binding Protein (FABP) comprising contacting the FABP with acompound having the structure:

whereinR₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅,        —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆,        -alykl-OR₁₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,        heteroaryl, or heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, aryl,        heteroaryl, or heterocyclyl;        when one of R₁ or R₂ is —C(═O)OH or —C(═O)OCH₃, then the other        of R₁ or R₂ is other than —C(═O)OR₁₃ where R₁₃ is alkyl,        heteroalkyl, substituted phenyl or benzyl, —C(═O)NHR₁₃R₁₄ where        one of R₁₃ or R₁₄ is —H, phenyl or substituted phenyl and the        other is —H, or —C(═O)NR₁₃R₁₄ where R₁₃ and R₁₄ combine to form        a piperidine or morpholine;        or a pharmaceutically acceptable salt thereof.

The present invention provides a method of identifying an agent thatinhibits the activity of a Fatty Acid Binding Protein (FABP) comprisingcontacting a Fatty Acid Binding Protein (FABP) expressed in the CNS withthe agent and separately with a compound having the structure

andcomparing the FABP inhibitory activity of the agent with the FABPinhibitory activity of the compound to identify an agent where FABPinhibitory activity is greater than that of the compound.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Scheme demonstrating anandamide inactivation and FABP drugtarget. Anandamide crosses the membrane by diffusion but requires FABPsfor transport through the cytoplasm to the endoplasmic reticulum forbreakdown by FAAH. FABP inhibitors prevent AEA from being delivered toFAAH for breakdown resulting in increased AEA levels at the receptor.

FIG. 2. Illustration of Footprint Similarity Score (a) reference ligand(b) comparing docked molecule with reference.

FIG. 3. Sequence alignment of FABP7 (light grey) and FABP5 (dark gray).Binding motif ARG106, ARG126, TYR128 is identical between FABP7 andFABP5 (dark gray). MET115 in FABP7 is similar but not identical toVAL115 in FABP5 (boxed). Oleic acid is encapsulated by the FABP.

FIG. 4. A flow chart describing the discovery of ligands through virtualscreening, biological assay, and medicinal chemistry.

FIG. 5. Lead compounds identified through the high throughputfluorescence displacement assay with NBD-stearate. Above is FABP7 witholeic acid bound and the molecular surface of oleic acid rendered ingrey. Each of the leads is represented in 3D (left) and 2D space(right). Shown in the 3D structures, each of the leads determined bybiological assay contained a carboxylate that bound to ARG106, ARG126,TYR128 a similar binding motif of the reference oleic acid.Additionally, each lead is shown to occupy the same chemical space asthe reference oleic acid.

FIG. 6. The VDW and ES footprint overlay of oleic acid and Compound 26.A significant VDW clash between the reference oleic acid and MET115 canbe seen in the VDW footprint. This steric clash is seen to be offset bythe strong ES interactions at ARG 106, ARG126, and TYR 128 seen in theES footprint. Energetically DOCK selected ligands from the ChemDivlibrary that did not have unfavorable interaction, both VDW and ES,intrinsically selecting inhibitors with a good ES footprint match whilemitigating the VDW clash at MET115.

FIG. 7. Compound 26 docked to FABP7 with key interactions seen withamino acid residues ARG126, TYR129, and ARG106. MET115 provides someselectivity by showing VDW clashing with the substrate Oleic Acid whichis a known FABP7 substrate.

FIG. 8. Binding analysis of Compound 26 (α-truxillic acid 1-naphthylester), Compound 49 (γ-truxillic acid 1-naphthyl ester), and BMS309403.(A) Assay in triplicate shows that Compound 26 attains a K_(i) withinnanoMolar ranges. (B) Compound 49 is as potent as BMS309403. (C)BMS309403 was found to be slightly more potent in this study (Ki, 0.75μM) than published (Ki, 0.89 μM), but still within range of this value.

FIG. 9. Compound 26 inhibits the cellular uptake of AEA. (A) AEA uptakein wild-type HeLa (un-shaded) or FABP5 shRNA-expressing HeLa (shaded)cells in the presence or absence of Compound 26. (B) AEA hydrolysis inFAAH-transfected HeLa cells in the presence or absence of Compound 26 orthe FAAH inhibitor URB597. **, p<0.01; ***, p<0.001 (n=3).

FIG. 10. Antinociceptive effects of Compound 26. (A) Compound 26 (20mg/kg, i.p.) reduces pain associated with the first phase (left panel)but not the second phase (right panel) of the formalin test. *, p<0.05(n=6). (B) Compound 26 (20 mg/kg, i.p.) alleviates carrageenan-inducedthermal hyperalgesia in mice. Concurrent administration of rimonabantand SR144528 (SR1/SR2) blocked the antinociceptive effects of Compound26. **, p<0.01 versus carrageenan-injected animals; ##, p<0.01 versusSR1/SR2-treated animals (n=6-9). (C) Compound 26 (20 mg/kg, i.p.)reduces carrageenan-induced paw edema. *, p<0.05 (n=6-9).

FIG. 11. Compound 26 is a weak agonist at PPARα and PPARγ receptors. (A)PPARα activation by Compound 26 and the PPARα agonist GW7647. (B)Activation of PPARγ receptors by Compound 26 compared to the PPARγagonist rosiglitazone (n=3).

FIG. 12. High throughput fluorescence displacement assay withNBD-stearate. Shown in blue is the NBD-stearate FABP5 complex with noinhibitor, in black is arachidonic acid a potent inhibitor of FABP5, andin red are the four lead compounds.

FIG. 13. Verification of high throughput fluorescent displacement assayresults. (A) Replicate testing of the lead compounds shows that Compound26 exhibits the best inhibition of FABP5. (B) Controls show that all ofthe lead compounds exhibited no detectable fluorescence in the assay nordid they add significantly to the fluorescence of the NBD-stearateprobe. *, p<0.05; **, p<0.01; ***, p<0.001 (n=3).

FIG. 14. Published in-vitro results of BMS480404. Highlighted in red isthe essential functional group require for binding. The trend appears tobe alpha keto acid >carboxylic acid >alpha hydroxy acid.

FIG. 15. Proposed binding of BMS480404-5 with water mediated hydrogenbonding between the alpha oxygen and ARG106 through water mediatedhydrogen bonding.

FIG. 16. Effects of FABP inhibitors upon nociception in mice. (A)Compounds 26 and 54 (20 mg/kg, i.p.) reduced carrageenan-induced thermalhyperalgesia (left panel) and paw edema (right panel) in mice.Measurements were performed 24 hrs after inhibitor administration. *,p<0.05; **, p<0.01. (B) Compounds 26 and 54 reduce the first (leftpanel) and second phases (right panel) of formalin-induced nociceptionin mice. *, p<0.05; **, p<0.01. (C) Compound 26 reduces aceticacid-induced writhing in mice. **, p<0.01. (D) Dose-response of Compound26-mediated inhibition of acetic acid writhing in mice. **, p<0.01. (E)The antinociceptive effects of Compound 26 are reversed by thecannabinoid receptor 1 antagonist SR141716 (SR1, 3 mg/kg) and theperoxisome proliferator-activated receptor alpha antagonist GW6471 (4mg/kg). *, p<0.05; **, p<0.01 versus control. ##, p<0.01; ###, p<0.001versus Compound 26 treated mice.

FIG. 17. Pharmacokinetic properties. Compound 26 elevates brain levelsof the endocannabinoid anandamide (AEA). *, p<0.05.

FIG. 18. Pharmacokinetic properties. Time course of Compound 26 inplasma (left panel) and brain (right panel) following a single injection(20 mg/kg, i.p.).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound having the structure:

whereinR₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅,        —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, alkyl-NR₁₅R₁₆, -alykl-OR₁₅, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or        heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl,        cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl;        when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₇, R₈, R₉,        R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other        than —C(═O)OR₁₃ where R₁₃ is —CH₃, —CH₂CH₃, tolyl or propyl        1-bromo-1-methylpropanoyloxybutyl ester, or —C(═O)NR₁₃R₁₄ where        one of R₁₃ or R₁₄ is phenyl and the other is —H, or both of R₁₃        and R₁₄ are —H; and        when one of R₁ or R₂ is —C(═O) OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈,        R₉, R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is        other than —C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the        other is (2-methylmercapto)phenyl;        or an enantiomer or pharmaceutically acceptable salt thereof.

In some embodiments, a compound having the structure:

whereinR₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ -alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅,        —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, alkyl-NR₁₅R₁₆, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl,        cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl;        when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₂, R₈, R₉,        R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other        than —C(═O)OR₁₃ where R₁₃ is —CH₃, —CH₂CH₃, —(CH₂)₄CH₃,        (CH₂)₇CH₃, —CH₂(CH₃)₂, —CH₂C(O) CH₃, tolyl, 1-Naphthol or propyl        1-bromo-1-methylpropanoyloxybutyl ester, or —C(═O)NR₁₃R₁₄ where        one of R₁₃ or R₁₄ is phenyl and the other is —H, or both of R₁₃        and R₁₄ are —H; and        when one of R₁ or R₂ is —C(═O) OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈,        R₉, R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is        other than —C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the        other is (2-methylmercapto)phenyl;        or an enantiomer or pharmaceutically acceptable salt thereof.

In some embodiments, a compound having the structure:

whereinR₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NR₁₅R₁₆, —SR₁₅, —SO₂R₁₅,        —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆, -alykl-OR₁₅,        C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or        heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl,        cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl;        when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₇, R₈, R₉,        R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other        than —C(═O)OR₁₃ where R₁₃ is —CH₂CH₃ or propyl        1-bromo-1-methylpropanoyloxybutyl ester;        when one of R₁ or R₂ is —C(═O) OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈,        R₉, R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is        other than —C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the        other is (2-methylmercapto)phenyl;        or an enantiomer or pharmaceutically acceptable salt thereof.

In some embodiments, a compound having the structure:

whereinR₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-NR₁₃R₁₄, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)NR₁₃R₁₄,-alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄, —C(—OH)C(═O)OR₁₃,—C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅,        —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆,        -alykl-OR₁₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,        heteroaryl, or heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl,        cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl;        when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₇, R₈, R₉,        R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other        than —C(═O)OR₁₃ where R₁₃ is —CH₃ or tolyl or —C(═O)NR₁₃R₁₄        where both of R₁₃ and R₁₄ are —H;        or an enantiomer or pharmaceutically acceptable salt thereof.

In some embodiments, the compound wherein

-   -   one of R₁ or R₂ is —C(═O)R₁₃, —C(═O)OR₁₃, or —C(═O)NHR₁₃,        -   wherein R₁₃ is aryl or heteroaryl; and    -   the other of R₁ or R₂ is —C(═O)OR₁₃,        -   wherein R₁₃ is H.

In some embodiments, the compound wherein

-   -   one of R₁ or R₂ is

-   -   and    -   the other of R₁ or R₂ is —C(═O)OH.

In some embodiments, the compound wherein

-   -   one of R₁ or R₂ is -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃,        -alkyl-C(═O)NHR₁₃, -alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃,        -alkyl-OR₁₃, -alkyl-NHR₁₃,        -   wherein R₁₃ is H, aryl or heteroaryl; and    -   the other of R₁ or R₂ is —C(═O)OR₁₃,        -   wherein R₁₃ is H.

In some embodiments, the compound wherein

-   -   one of R₁ or R₂ is

and

-   -   the other of R₁ or R₂ is —C(═O)OH.

In some embodiments, the compound wherein

-   -   one of R₁ or R₂ is -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃,        -alkyl-C(═O)NHR₁₃, -alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃,        -alkyl-OR₁₃, -alkyl-NHR₁₃,        -   wherein R₁₃ is H, aryl or heteroaryl; and    -   the other of R₁ or R₂ is —C(—OH)C(═O)OR₁₃, or —C(═O) C(═O)OR₁₃,        -   wherein R₁₃ is H or alkyl.

In some embodiments, the compound wherein

-   -   one of R₁ or R₂ is

and

-   -   the other of R₁ or R₂ is

In some embodiments, the compound wherein

-   -   R₁₃ is

-   -   wherein each of X, Y, X are independently, H, halogen, —NO₂,        —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅, —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃,        -alkyl-NR₁₅R₁₆, -alykl-OR₁₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀        alkynyl, aryl, heteroaryl, or heterocyclyl;        -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀            alkyl, C₂₋₁₀ C₂₋₁₀ alkenyl, alkynyl, heteroalkyl,            cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl;

In some embodiments, the compound having the structure:

In some embodiments, the compound having the structure:

In some embodiments, the invention provides a compound having thestructure:

or an enantiomer or pharmaceutically acceptable salt thereof.

The present invention provides a process for producing the compound ofthe present invention comprising:

-   -   (a) contacting a compound having the structure:

-   -   with acetic anhydride in the presence of sodium acetate so as to        produce a compound having the structure:

-   -   (b) reacting the product of step (a) with a nucleophile (Nuc) in        a first suitable solvent in the presence of an amine base so as        to produce a mixture of enantiomers having the structures:

In some embodiments, the method wherein the nucleophile used in step (b)is

In some embodiments, the method wherein the nucleophile used in step (b)is a chiral nucleophile.

In some embodiments, the method wherein the nucleophile used in step (b)is (S)-(−)-1-phenylethanol.

In some embodiments, the method further comprising separating thediastereomeric products of step (b).

In some embodiments, the method wherein the products of step (b) are

In some embodiments, the method further comprising

-   -   (c) separating the diastereomeric products of step (b) to        produce enantiopure compounds having the structure:

-   -   (d) reacting a product of step (c) with a coupling reagent in        the presence of a nucleophile in a second suitable solvent so as        to produce enantiopure compounds having the structure:

-   -   (e) reacting the product of step (d) with hydrogen in the        presence of palladium on carbon to produce an enantiopure        compound having the structure:

In some embodiments, the method wherein the nucleophile used in step (d)is

In some embodiments of the above process, the compound produced has thestructure:

or an enantiomer thereof.

The present invention provides a method of inhibiting the activity of aFatty Acid Binding Protein (FABP) comprising contacting the FABP with acompound having the structure:

whereinR₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅,        —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆,        C₁₋₁₀ alkyl, C₂₋₄₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or        heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, aryl,        heteroaryl, or heterocyclyl;        when one of R₁ or R₂ is —C(═O)OH or —C(═O)OCH₃, then the other        of R₁ or R₂ is other than —C(═O) OR₁₃ where R₁₃ is alkyl,        heteroalkyl, substituted phenyl or benzyl, —C(═O)NHR₁₃R₁₄ where        one of R₁₃ or R₁₄ is —H, phenyl or substituted phenyl and the        other is —H, or —C(═O)NR₁₃R₁₄ where R₁₃ and R₁₄ combine to form        a piperidine or morpholine;        or a pharmaceutically acceptable salt thereof.

In some embodiments of the method of inhibiting the activity of a FattyAcid Binding Protein (FABP), wherein when one of R₁ or R₂ is —C(═O) OHor —C(═O) OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are eachH, then the other of R₁ or R₂ is other than —C(═O) OR₁₃ where R₁₃ isalkyl, heteroalkyl, substituted phenyl or benzyl, —C(═O)NHR₁₃R₁₄ whereone of R₁₃ or R₁₄ is —H, phenyl or substituted phenyl and the other is—H, or —C(═O)NR₁₃R₁₄ where R₁₃ and R₁₄ combine to form a piperidine ormorpholine.

In some embodiments of the method of inhibiting the activity of a FattyAcid Binding Protein (FABP), wherein the compound has the structure:

whereinR₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₂, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅,        —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NR₁₅R₁₆, -alykl-OR₁₅, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or        heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl,        cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl;        when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₂, R₈, R₉,        R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other        than —C(═O)OR₁₃ where R₁₃ is —CH₃, —CH₂CH₃, —(CH₂)₄CH₃,        —(CH₂)₇CH₃, —CH₂(CH₃)₂, —CH₂C(O)CH₃, tolyl, 1-Naphthol or propyl        1-bromo-1-methylpropanoyloxybutyl ester, or —C(═O)NR₁₃R₁₄ where        one of R₁₃ or R₁₄ is phenyl and the other is —H, or both of R₁₃        and R₁₄ are —H; and        when one of R₁ or R₂ is —C(═O) OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈,        R₉, R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is        other than —C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the        other is (2-methylmercapto)phenyl;        or an enantiomer or pharmaceutically acceptable salt thereof.

In some embodiments of the method of inhibiting the activity of a FattyAcid Binding Protein (FABP), wherein the compound has the structure:

whereinR₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅,        —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆,        -alykl-OR₁₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,        heteroaryl, or heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl,        cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl;        when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₇, R₈, R₉,        R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other        than —C(═O)OR₁₃ where R₁₃ is —CH₃, —CH₂CH₃, tolyl or propyl        1-bromo-1-methylpropanoyloxybutyl ester, or —C(═O)NR₁₃R₁₄ where        one of R₁₃ or R₁₄ is phenyl and the other is —H, or both of R₁₃        and R₁₄ are —H; and        when one of R₁ or R₂ is —C(═O) OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈,        R₉, R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is        other than —C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the        other is (2-methylmercapto)phenyl;        or an enantiomer or pharmaceutically acceptable salt thereof.

In some embodiments of any of the above methods, the compound having thestructure:

or an enantiomer or pharmaceutically acceptable salt thereof.

In some embodiments of the method of inhibiting the activity of a FattyAcid Binding Protein (FABP), wherein the compound inhibits binding of anFABP ligand to the FABP.

In some embodiments of the method of inhibiting the activity of a FattyAcid Binding Protein (FABP), wherein the FABP ligand is anendocannabinoid ligand.

In some embodiments of the method of inhibiting the activity of a FattyAcid Binding Protein (FABP), wherein the FABP ligand is anandamide (AEA)or 2-arachidonoylglycerol (2-AG).

The present invention provides a method of identifying an agent thatinhibits the activity of a Fatty Acid Binding Protein (FABP) comprisingcontacting a Fatty Acid Binding Protein (FABP) expressed in the CNS withthe agent and separately with a compound having the structure

andcomparing the FABP inhibitory activity of the agent with the FABPinhibitory activity of the compound to identify an agent where FABPinhibitory activity is greater than that of the compound.

In some embodiments, a method of inhibiting the activity of a Fatty AcidBinding Protein (FABP) comprising contacting the FABP with a compoundhaving the structure:

whereinR₁ and R₂ are each independently —C(═O)R₁₃, —C(═O)OR₁₃, —C(═O)NR₁₃R₁₄,-alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O) C(═O)OR₁₃ or —C═C—R₁₃,

-   -   wherein R₁₃ and R₁₄ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloalkyl,        aryl, heteroaryl, heterocyclyl or combine to form a cycloalkyl        or heterocyclyl;        R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each        independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆, —SR₁₅,        —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆,        -alykl-OR₁₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,        heteroaryl, or heterocyclyl;    -   wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀        alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, aryl,        heteroaryl, or heterocyclyl;        or a pharmaceutically acceptable salt thereof.

The following compounds are available from ChemDiv (San Diego, Calif.,USA):

ChemDiv ID Structure Number

8009- 2334

8009- 3434

8010- 1995

8010- 1996

8010- 1997

8008- 6749

8008- 8584

In some embodiments, a method of treating a neurological disorderaffects at least one of movement, memory, mood, appetite, nociception,endocrine regulation, thermoregulation, sensory perception, or cognitivefunctions.

In some embodiments, a method of treating a neurological disorderassociated with drug addiction, depression, compulsive behavior,neuropathic pain, or a movement disorder.

In some embodiments, a method of treating drug addiction, depression,compulsive behavior, neuropathic pain, inflammatory pain, or a movementdisorder.

In some embodiments, a method of treating pain, neuropathic pain, orinflammatory pain.

As used herein, the term “endocannabinoid” includes any molecule thatactivates cannabinoid receptors. Examples of such receptors are CB1 andCB2. Examples of endocannabinoids are arachidonoyl ethanolamide (AEA)and 2-arachidonoyl glycerol (2-AG).

As used herein, the term “fatty acid binding protein” or “FABP” refersto fatty acid binding proteins (FABPs) that function as intracellularcarriers that shuttle cannabinoids (and by extension fatty acid amides(FAAs)) to FAAH where cannabinoids are hydrolyzed and degraded. Further,uptake of endocannabinoids (and by extension FAAs) by the cell and thesubsequent hydrolysis of endocannabinoids (and by extension FAAs) areenhanced by FABPs, and inhibiting the interaction of endocannabinoids(and by extension FAAs) with FABPs reduces endocannabinoid (and byextension FAA) uptake and hydrolysis. FABPS include, for example, fattyacid binding protein 1 (FABP 1), fatty acid binding protein 2 (FABP 2),fatty acid binding protein 3 (FABP 3), fatty acid binding protein 4(FABP 4), fatty acid binding protein 5 (FABP 5), fatty acid bindingprotein 6 (FABP 6), fatty acid binding protein 7 (FABP 7), fatty acidbinding protein 8 (FABP 8), fatty acid binding protein 9 (FABP 9), fattyacid binding protein 10 (FABP 10), fatty acid binding protein 11 (FABP11), fatty acid binding protein 5-like (FABP 5-like 1), fatty acidbinding protein 5-like 2 (FABP 5-like 2), fatty acid binding protein5-like 3 (FABP 5-like 3), fatty acid binding protein 5-like 4 (FABP5-like 4), fatty acid binding protein 5-like 5 (FABP 5-like 5), fattyacid binding protein 5-like 6 (FABP 5-like 6), and fatty acid bindingprotein 5-like 7 (FABP 5-like 7) (see Chmurzynska et al. 2006 and PCTInternational Application Publication No. WO 2010/083532 A1, thecontents of each of which are hereby incorporated by reference).

As used herein, the term “therapeutic agent” refers to any agent used totreat a disease or that provides a beneficial therapeutic effect to asubject.

As used herein, the phrase “inhibits the interaction” is employed hereinto refer to any disruption, partial or total, of the natural effect ofFABPs on the metabolism of endocannabinoids.

As used herein, the term “activity” refers to the activation,production, expression, synthesis, intercellular effect, and/orpathological or aberrant effect of the referenced molecule, eitherinside and/or outside of a cell. Such molecules include, but are notlimited to, cytokines, enzymes, growth factors, pro-growth factors,active growth factors, and pro-enzymes. Molecules such as cytokines,enzymes, growth factors, pro-growth factors, active growth factors, andpro-enzymes may be produced, expressed, or synthesized within a cellwhere they may exert an effect. Such molecules may also be transportedoutside of the cell to the extracellular matrix where they may induce aneffect on the extracellular matrix or on a neighboring cell. It isunderstood that activation of inactive cytokines, enzymes andpro-enzymes may occur inside and/or outside of a cell and that bothinactive and active forms may be present at any point inside and/oroutside of a cell. It is also understood that cells may possess basallevels of such molecules for normal function and that abnormally high orlow levels of such active molecules may lead to pathological or aberranteffects that may be corrected by pharmacological intervention.

As used herein, “treating” means reducing, slowing, stopping,preventing, reversing, or in any way improving the progression of adisease or disorder or a symptom of the disease or disorder.

In some embodiments, the compounds of the present invention include allhydrates, solvates, and complexes of the compounds used by thisinvention.

In some embodiments, if a chiral center or another form of an isomericcenter is present in a compound of the present invention, all forms ofsuch isomer or isomers, including enantiomers and diastereomers, areintended to be covered herein.

In some embodiments, if a chiral center or another form of an isomericcenter is present in a compound of the present invention, onlyenantiomeric forms are intended to be covered herein.

Compounds containing a chiral center may be used as a racemic mixture,an enantiomerically enriched mixture, or the racemic mixture may beseparated using well-known techniques and an individual enantiomer maybe used alone. The compounds described in the present invention are inracemic form or as individual enantiomers.

As used herein, “enantiomers” are non-identical, non-superimposiblemirror images of each other. For any given chiral compound, only onepair of enantiomers exists. The enantiomers can be separated using knowntechniques, including those described in Pure and Applied Chemistry 69,1469-1474, (1997) IUPAC.

In cases in which compounds have unsaturated carbon-carbon double bonds,both the cis (Z) and trans (E) isomers are within the scope of thisinvention.

The compounds of the subject invention may have spontaneous tautomericforms. In cases wherein compounds may exist in tautomeric forms, such asketo-enol tautomers, each tautomeric form is contemplated as beingincluded within this invention whether existing in equilibrium orpredominantly in one form.

In the compound structures depicted herein, hydrogen atoms are not shownfor carbon atoms having less than four bonds to non-hydrogen atoms.However, it is understood that enough hydrogen atoms exist on saidcarbon atoms to satisfy the octet rule.

This invention also provides isotopic variants of the compoundsdisclosed herein, including wherein the isotopic atom is ²H and/orwherein the isotopic atom ¹³C. Accordingly, in the compounds providedherein hydrogen can be enriched in the deuterium isotope. It is to beunderstood that the invention encompasses all such isotopic forms.

It is understood that the structures described in the embodiments of themethods hereinabove can be the same as the structures of the compoundsdescribed hereinabove.

It is understood that where a numerical range is recited herein, thepresent invention contemplates each integer between, and including, theupper and lower limits, unless otherwise stated.

Except where otherwise specified, if the structure of a compound of thisinvention includes an asymmetric carbon atom, it is understood that thecompound occurs as a racemate, racemic mixture, and isolated singleenantiomer. All such isomeric forms of these compounds are expresslyincluded in this invention. Except where otherwise specified, eachstereogenic carbon may be of the R or S configuration. It is to beunderstood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis, such as those described in“Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S.Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution maybe carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include all isotopes of atomsoccurring on the compounds disclosed herein. Isotopes include thoseatoms having the same atomic number but different mass numbers.

By way of general example and without limitation, isotopes of hydrogeninclude tritium and deuterium. Isotopes of carbon include C-13 and C-14.

It will be noted that any notation of a carbon in structures throughoutthis application, when used without further notation, are intended torepresent all isotopes of carbon, such as ¹²C, ¹³C, or ¹⁴C. Furthermore,any compounds containing ¹³C or ¹⁴C may specifically have the structureof any of the compounds disclosed herein.

It will also be noted that any notation of a hydrogen in structuresthroughout this application, when used without further notation, areintended to represent all isotopes of hydrogen, such as ¹H, ²H, or ³H.Furthermore, any compounds containing ²H or ³H may specifically have thestructure of any of the compounds disclosed herein.

Isotopically-labeled compounds can generally be prepared by conventionaltechniques known to those skilled in the art using appropriateisotopically-labeled reagents in place of the non-labeled reagentsemployed.

In the compounds used in the method of the present invention, thesubstituents may be substituted or unsubstituted, unless specificallydefined otherwise.

In the compounds used in the method of the present invention, alkyl,heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groupscan be further substituted by replacing one or more hydrogen atoms withalternative non-hydrogen groups. These include, but are not limited to,halo, hydroxy, mercapto, amino, carboxy, cyano, carbamoyl andaminocarbonyl and aminothiocarbonyl.

It is understood that substituents and substitution patterns on thecompounds used in the method of the present invention can be selected byone of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art from readily available starting materials. If asubstituent is itself substituted with more than one group, it isunderstood that these multiple groups may be on the same carbon or ondifferent carbons, so long as a stable structure results.

In choosing the compounds used in the method of the present invention,one of ordinary skill in the art will recognize that the varioussubstituents, i.e. R₁, R₂, etc. are to be chosen in conformity withwell-known principles of chemical structure connectivity.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and may be unsubstituted or substituted. Thus, C₁-C_(n) asin “C₁-C_(n) alkyl” is defined to include individual groups each having1, 2, n-1 or n carbons in a linear or branched arrangement. For example,C₁-C₆, as in “C₁-C₆ alkyl” is defined to include individual groups eachhaving 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement,and specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, pentyl, hexyl, and octyl.

As used herein, “alkenyl” refers to a non-aromatic hydrocarbon radical,straight or branched, containing at least 1 carbon to carbon doublebond, and up to the maximum possible number of non-aromaticcarbon-carbon double bonds may be present, and may be unsubstituted orsubstituted. For example, “C₂-C₆ alkenyl” means an alkenyl radicalhaving 2, 3, 4, 5, or 6 carbon atoms, and up to 1, 2, 3, 4, or 5carbon-carbon double bonds respectively. Alkenyl groups include ethenyl,propenyl, butenyl and cyclohexenyl.

The term “alkynyl” refers to a hydrocarbon radical straight or branched,containing at least 1 carbon to carbon triple bond, and up to themaximum possible number of non-aromatic carbon-carbon triple bonds maybe present, and may be unsubstituted or substituted. Thus, “C₂-C₆alkynyl” means an alkynyl radical having 2 or 3 carbon atoms and 1carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2carbon-carbon triple bonds, or having 6 carbon atoms and up to 3carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl andbutynyl.

“Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, adivalent alkane, alkene and alkyne radical, respectively. It isunderstood that an alkylene, alkenylene, and alkynylene may be straightor branched. An alkylene, alkenylene, and alkynylene may beunsubstituted or substituted.

As used herein, “heteroalkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and at least 1 heteroatom within the chain or branch.

As used herein, “heterocycle” or “heterocyclyl” as used herein isintended to mean a 5- to 10-membered nonaromatic ring containing from 1to 4 heteroatoms selected from the group consisting of O, N and S, andincludes bicyclic groups. “Heterocyclyl” therefore includes, but is notlimited to the following: imidazolyl, piperazinyl, piperidinyl,pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl,dihydropiperidinyl, tetrahydrothiophenyl and the like. If theheterocycle contains a nitrogen, it is understood that the correspondingN-oxides thereof are also encompassed by this definition.

As herein, “cycloalkyl” shall mean cyclic rings of alkanes of three toeight total carbon atoms, or any number within this range (i.e.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl orcyclooctyl).

As used herein, “monocycle” includes any stable polyatomic carbon ringof up to 10 atoms and may be unsubstituted or substituted. Examples ofsuch non-aromatic monocycle elements include but are not limited to:cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of sucharomatic monocycle elements include but are not limited to: phenyl.

As used herein, “bicycle” includes any stable polyatomic carbon ring ofup to 10 atoms that is fused to a polyatomic carbon ring of up to atomswith each ring being independently unsubstituted or substituted.Examples of such non-aromatic bicycle elements include but are notlimited to: decahydronaphthalene. Examples of such aromatic bicycleelements include but are not limited to: naphthalene.

As used herein, “aryl” is intended to mean any stable monocyclic,bicyclic or polycyclic carbon ring of up to 10 atoms in each ring,wherein at least one ring is aromatic, and may be unsubstituted orsubstituted. Examples of such aryl elements include phenyl, p-toluenyl(4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl,phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituentis bicyclic and one ring is non-aromatic, it is understood thatattachment is via the aromatic ring.

As used herein, the term “polycyclic” refers to unsaturated or partiallyunsaturated multiple fused ring structures, which may be unsubstitutedor substituted.

The term “alkylryl” refers to alkyl groups as described above whereinone or more bonds to hydrogen contained therein are replaced by a bondto an aryl group as described above. It is understood that an“arylalkyl” group is connected to a core molecule through a bond fromthe alkyl group and that the aryl group acts as a substituent on thealkyl group. Examples of arylalkyl moieties include, but are not limitedto, benzyl (phenylmethyl),p-trifluoromethylbenzyl(4-trifluoromethylphenylmethyl), 1-phenylethyl,2-phenylethyl, 3-phenylpropyl, 2-phenylpropyl and the like.

The term “heteroaryl”, as used herein, represents a stable monocyclic,bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein atleast one ring is aromatic and contains from 1 to 4 heteroatoms selectedfrom the group consisting of O, N and S. Bicyclic aromatic heteroarylgroups include phenyl, pyridine, pyrimidine or pyridizine rings that are(a) fused to a 6-membered aromatic (unsaturated) heterocyclic ringhaving one nitrogen atom; (b) fused to a 5- or 6-membered aromatic(unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused toa 5-membered aromatic (unsaturated) heterocyclic ring having onenitrogen atom together with either one oxygen or one sulfur atom; or (d)fused to a 5-membered aromatic (unsaturated) heterocyclic ring havingone heteroatom selected from O, N or S. Heteroaryl groups within thescope of this definition include but are not limited to:benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl,benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl,cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl,isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl,naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline,oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl,pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl,thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl,hexahydroazepinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl,carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl,benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl,furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where theheteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively. Ifthe heteroaryl contains nitrogen atoms, it is understood that thecorresponding N-oxides thereof are also encompassed by this definition.

The term “alkylheteroaryl” refers to alkyl groups as described abovewherein one or more bonds to hydrogen contained therein are replaced bya bond to an heteroaryl group as described above. It is understood thatan “alkylheteroaryl” group is connected to a core molecule through abond from the alkyl group and that the heteroaryl group acts as asubstituent on the alkyl group. Examples of alkylheteroaryl moietiesinclude, but are not limited to, —CH₂—(C₅H₄N), —CH₂—CH₂—(C₅H₄N) and thelike.

The term “heterocycle” or “heterocyclyl” refers to a mono- or polycyclicring system which can be saturated or contains one or more degrees ofunsaturation and contains one or more heteroatoms. Preferred heteroatomsinclude N, O, and/or S, including N-oxides, sulfur oxides, and dioxides.Preferably the ring is three to ten-membered and is either saturated orhas one or more degrees of unsaturation. The heterocycle may beunsubstituted or substituted, with multiple degrees of substitutionbeing allowed. Such rings may be optionally fused to one or more ofanother “heterocyclic” ring(s), heteroaryl ring(s), aryl ring(s), orcycloalkyl ring(s). Examples of heterocycles include, but are notlimited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane,piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine,tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.

The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclylsubstituents may be substituted or unsubstituted, unless specificallydefined otherwise. In the compounds of the present invention, alkyl,alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl groups can befurther substituted by replacing one or more hydrogen atoms withalternative non-hydrogen groups. These include, but are not limited to,halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

As used herein, the term “halogen” refers to F, Cl, Br, and I.

The terms “substitution”, “substituted” and “substituent” refer to afunctional group as described above in which one or more bonds to ahydrogen atom contained therein are replaced by a bond to non-hydrogenor non-carbon atoms, provided that normal valencies are maintained andthat the substitution results in a stable compound. Substituted groupsalso include groups in which one or more bonds to a carbon(s) orhydrogen(s) atom are replaced by one or more bonds, including double ortriple bonds, to a heteroatom. Examples of substituent groups includethe functional groups described above, and halogens (i.e., F, Cl, Br,and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl,n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, suchas methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such asphenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) andp-trifluoromethylbenzyloxy(4-trifluoromethylphenylmethoxy);heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl,methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto;sulfanyl groups, such as methylsulfanyl, ethylsulfanyl andpropylsulfanyl; cyano; amino groups, such as amino, methylamino,dimethylamino, ethylamino, and diethylamino; and carboxyl. Wheremultiple substituent moieties are disclosed or claimed, the substitutedcompound can be independently substituted by one or more of thedisclosed or claimed substituent moieties, singly or pluraly. Byindependently substituted, it is meant that the (two or more)substituents can be the same or different.

The term “tolyl” refers to one of the three CH₃C₆H₄— isomeric groupsderived from toluene.

The term “piperidine” refers to a heterocyclic amine consists of asix-membered ring containing five methylene units and one nitrogen atom.

The term “morpholine” refers to a heterocyclic amine consists of asix-membered ring containing four methylene units, one nitrogen atom andone oxygen atom, wherein there are two pairs of methylene units linkingthe nitrogen atom and oxygen atom.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

In choosing the compounds of the present invention, one of ordinaryskill in the art will recognize that the various substituents, i.e. R₁,R₂, etc. are to be chosen in conformity with well-known principles ofchemical structure connectivity. The various R groups attached to thearomatic rings of the compounds disclosed herein may be added to therings by standard procedures, for example those set forth in AdvancedOrganic Chemistry: Part B: Reaction and Synthesis, Francis Carey andRichard Sundberg, (Springer) 5th ed. Edition. (2007), the content ofwhich is hereby incorporated by reference.

The compounds used in the method of the present invention may beprepared by techniques well known in organic synthesis and familiar to apractitioner ordinarily skilled in the art. However, these may not bethe only means by which to synthesize or obtain the desired compounds.

The compounds used in the method of the present invention may beprepared by techniques described in Vogel's Textbook of PracticalOrganic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Furnis, A. J.Hannaford, P. W. G. Smith, (Prentice Hall) 5^(th) Edition (1996),March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5^(th)Edition (2007), and references therein, which are incorporated byreference herein. However, these may not be the only means by which tosynthesize or obtain the desired compounds.

Another aspect of the invention comprises a compound used in the methodof the present invention as a pharmaceutical composition.

In some embodiments, a pharmaceutical composition comprising thecompound of the present invention and a pharmaceutically acceptablecarrier.

As used herein, the term “pharmaceutically active agent” means anysubstance or compound suitable for administration to a subject andfurnishes biological activity or other direct effect in the treatment,cure, mitigation, diagnosis, or prevention of disease, or affects thestructure or any function of the subject. Pharmaceutically active agentsinclude, but are not limited to, substances and compounds described inthe Physicians' Desk Reference (PDR Network, LLC; 64th edition; Nov. 15,2009) and “Approved Drug Products with Therapeutic EquivalenceEvaluations” (U.S. Department Of Health And Human Services, 30^(th)edition, 2010), which are hereby incorporated by reference.Pharmaceutically active agents which have pendant carboxylic acid groupsmay be modified in accordance with the present invention using standardesterification reactions and methods readily available and known tothose having ordinary skill in the art of chemical synthesis. Where apharmaceutically active agent does not possess a carboxylic acid group,the ordinarily skilled artisan will be able to design and incorporate acarboxylic acid group into the pharmaceutically active agent whereesterification may subsequently be carried out so long as themodification does not interfere with the pharmaceutically active agent'sbiological activity or effect.

The compounds used in the method of the present invention may be in asalt form. As used herein, a “salt” is a salt of the instant compoundswhich has been modified by making acid or base salts of the compounds.In the case of compounds used to treat an infection or disease caused bya pathogen, the salt is pharmaceutically acceptable. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as phenols. The salts can bemade using an organic or inorganic acid. Such acid salts are chlorides,bromides, sulfates, nitrates, phosphates, sulfonates, formates,tartrates, maleates, malates, citrates, benzoates, salicylates,ascorbates, and the like. Phenolate salts are the alkaline earth metalsalts, sodium, potassium or lithium. The term “pharmaceuticallyacceptable salt” in this respect, refers to the relatively non-toxic,inorganic and organic acid or base addition salts of compounds of thepresent invention. These salts can be prepared in situ during the finalisolation and purification of the compounds of the invention, or byseparately reacting a purified compound of the invention in its freebase or free acid form with a suitable organic or inorganic acid orbase, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The compounds of the present invention may also form salts with basicamino acids such a lysine, arginine, etc. and with basic sugars such asN-methylglucamine, 2-amino-2-deoxyglucose, etc. and any otherphysiologically non-toxic basic substance.

The compounds used in the method of the present invention may beadministered in various forms, including those detailed herein. Thetreatment with the compound may be a component of a combination therapyor an adjunct therapy, i.e. the subject or patient in need of the drugis treated or given another drug for the disease in conjunction with oneor more of the instant compounds. This combination therapy can besequential therapy where the patient is treated first with one drug andthen the other or the two drugs are given simultaneously. These can beadministered independently by the same route or by two or more differentroutes of administration depending on the dosage forms employed.

As used herein, a “pharmaceutically acceptable carrier” is apharmaceutically acceptable solvent, suspending agent or vehicle, fordelivering the instant compounds to the animal or human. The carrier maybe liquid or solid and is selected with the planned manner ofadministration in mind. Liposomes are also a pharmaceutically acceptablecarrier as are slow-release vehicles.

The dosage of the compounds administered in treatment will varydepending upon factors such as the pharmacodynamic characteristics of aspecific chemotherapeutic agent and its mode and route ofadministration; the age, sex, metabolic rate, absorptive efficiency,health and weight of the recipient; the nature and extent of thesymptoms; the kind of concurrent treatment being administered; thefrequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds used in the method of the presentinvention may comprise a single compound or mixtures thereof withadditional antitumor agents. The compounds can be administered in oraldosage forms as tablets, capsules, pills, powders, granules, elixirs,tinctures, suspensions, syrups, and emulsions. The compounds may also beadministered in intravenous (bolus or infusion), intraperitoneal,subcutaneous, or intramuscular form, or introduced directly, e.g. byinjection, topical application, or other methods, into or topically ontoa site of disease or lesion, all using dosage forms well known to thoseof ordinary skill in the pharmaceutical arts.

The compounds used in the method of the present invention can beadministered in admixture with suitable pharmaceutical diluents,extenders, excipients, or in carriers such as the novel programmablesustained-release multi-compartmental nanospheres (collectively referredto herein as a pharmaceutically acceptable carrier) suitably selectedwith respect to the intended form of administration and as consistentwith conventional pharmaceutical practices. The unit will be in a formsuitable for oral, nasal, rectal, topical, intravenous or directinjection or parenteral administration. The compounds can beadministered alone or mixed with a pharmaceutically acceptable carrier.This carrier can be a solid or liquid, and the type of carrier isgenerally chosen based on the type of administration being used. Theactive agent can be co-administered in the form of a tablet or capsule,liposome, as an agglomerated powder or in a liquid form. Examples ofsuitable solid carriers include lactose, sucrose, gelatin and agar.Capsule or tablets can be easily formulated and can be made easy toswallow or chew; other solid forms include granules, and bulk powders.Tablets may contain suitable binders, lubricants, diluents,disintegrating agents, coloring agents, flavoring agents, flow-inducingagents, and melting agents. Examples of suitable liquid dosage formsinclude solutions or suspensions in water, pharmaceutically acceptablefats and oils, alcohols or other organic solvents, including esters,emulsions, syrups or elixirs, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules and effervescentpreparations reconstituted from effervescent granules. Such liquiddosage forms may contain, for example, suitable solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, thickeners,and melting agents. Oral dosage forms optionally contain flavorants andcoloring agents. Parenteral and intravenous forms may also includeminerals and other materials to make them compatible with the type ofinjection or delivery system chosen.

Techniques and compositions for making dosage forms useful in thepresent invention are described in the following references: 7 ModernPharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979);Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel,Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976);Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company,Easton, Pa., 1985); Advances in Pharmaceutical Sciences (DavidGanderton, Trevor Jones, Eds., 1992); Advances in PharmaceuticalSciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds.,1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugsand the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs andthe Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in theBiological Sciences. Series in Pharmaceutical Technology; J. G. Hardy,S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and thePharmaceutical Sciences, Vol (Gilbert S. Banker, Christopher T. Rhodes,Eds.). All of the aforementioned publications are incorporated byreference herein.

Tablets may contain suitable binders, lubricants, disintegrating agents,coloring agents, flavoring agents, flow-inducing agents, and meltingagents. For instance, for oral administration in the dosage unit form ofa tablet or capsule, the active drug component can be combined with anoral, non-toxic, pharmaceutically acceptable, inert carrier such aslactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol,sorbitol and the like. Suitable binders include starch, gelatin, naturalsugars such as glucose or beta-lactose, corn sweeteners, natural andsynthetic gums such as acacia, tragacanth, or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes, and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride, and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compounds used in the method of the present invention may also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids suchas lecithin, sphingomyelin, proteolipids, protein-encapsulated vesiclesor from cholesterol, stearylamine, or phosphatidylcholines. Thecompounds may be administered as components of tissue-targetedemulsions.

The compounds used in the method of the present invention may also becoupled to soluble polymers as targetable drug carriers or as a prodrug.Such polymers include polyvinylpyrrolidone, pyran copolymer,polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyglycolicacid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

Gelatin capsules may contain the active ingredient compounds andpowdered carriers, such as lactose, starch, cellulose derivatives,magnesium stearate, stearic acid, and the like. Similar diluents can beused to make compressed tablets. Both tablets and capsules can bemanufactured as immediate release products or as sustained releaseproducts to provide for continuous release of medication over a periodof hours. Compressed tablets can be sugar-coated or film-coated to maskany unpleasant taste and protect the tablet from the atmosphere, orenteric coated for selective disintegration in the gastrointestinaltract.

For oral administration in liquid dosage form, the oral drug componentsare combined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Examples ofsuitable liquid dosage forms include solutions or suspensions in water,pharmaceutically acceptable fats and oils, alcohols or other organicsolvents, including esters, emulsions, syrups or elixirs, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules and effervescent preparations reconstituted from effervescentgranules. Such liquid dosage forms may contain, for example, suitablesolvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. In general, water, asuitableoil, saline, aqueous dextrose (glucose), and related sugar solutions andglycols such as propylene glycol or polyethylene glycols are suitablecarriers for parenteral solutions. Solutions for parenteraladministration preferably contain a water soluble salt of the activeingredient, suitable stabilizing agents, and if necessary, buffersubstances. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

The compounds used in the method of the present invention may also beadministered in intranasal form via use of suitable intranasal vehicles,or via transdermal routes, using those forms of transdermal skin patcheswell known to those of ordinary skill in that art. To be administered inthe form of a transdermal delivery system, the dosage administrationwill generally be continuous rather than intermittent throughout thedosage regimen.

Parenteral and intravenous forms may also include minerals and othermaterials such as solutol and/or ethanol to make them compatible withthe type of injection or delivery system chosen.

The compounds and compositions of the present invention can beadministered in oral dosage forms as tablets, capsules, pills, powders,granules, elixirs, tinctures, suspensions, syrups, and emulsions. Thecompounds may also be administered in intravenous (bolus or infusion),intraperitoneal, subcutaneous, or intramuscular form, or introduceddirectly, e.g. by topical administration, injection or other methods, tothe afflicted area, such as a wound, including ulcers of the skin, allusing dosage forms well known to those of ordinary skill in thepharmaceutical arts.

Specific examples of pharmaceutically acceptable carriers and excipientsthat may be used to formulate oral dosage forms of the present inventionare described in U.S. Pat. No. 3,903,297 to Robert, issued Sep. 2, 1975.Techniques and compositions for making dosage forms useful in thepresent invention are described-in the following references: 7 ModernPharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979);Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel,Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976);Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company,Easton, Pa., 1985); Advances in Pharmaceutical Sciences (DavidGanderton, Trevor Jones, Eds., 1992); Advances in PharmaceuticalSciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds.,1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugsand the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs andthe Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in theBiological Sciences. Series in Pharmaceutical Technology; J. G. Hardy,S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and thePharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T.Rhodes, Eds.). All of the aforementioned publications are incorporatedby reference herein.

The term “prodrug” as used herein refers to any compound that whenadministered to a biological system generates the compound of theinvention, as a result of spontaneous chemical reaction(s), enzymecatalyzed chemical reaction(s), photolysis, and/or metabolic chemicalreaction(s). A prodrug is thus a covalently modified analog or latentform of a compound of the invention.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, powders, and chewing gum; or in liquid dosageforms, such as elixirs, syrups, and suspensions, including, but notlimited to, mouthwash and toothpaste. It can also be administeredparentally, in sterile liquid dosage forms.

Solid dosage forms, such as capsules and tablets, may be enteric-coatedto prevent release of the active ingredient compounds before they reachthe small intestine. Materials that may be used as enteric coatingsinclude, but are not limited to, sugars, fatty acids, proteinaceoussubstances such as gelatin, waxes, shellac, cellulose acetate phthalate(CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetatesuccinate, hydroxy propyl methyl cellulose phthalate, hydroxy propylmethyl cellulose acetate succinate (hypromellose acetate succinate),polyvinyl acetate phthalate (PVAP), and methyl methacrylate-methacrylicacid copolymers.

The compounds and compositions of the invention can be coated ontostents for temporary or permanent implantation into the cardiovascularsystem of a subject.

Each embodiment disclosed herein is contemplated as being applicable toeach of the other disclosed embodiments. Thus, all combinations of thevarious elements described herein are within the scope of the invention.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details Materials and Methods

Those having ordinary skill in the art of organic synthesis willappreciate that modifications to general procedures and synthetic routescontained in this application can be used to yield additionalderivatives and structurally diverse compounds. Suitable organictransformations are described in in March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure (Wiley-Interscience; 6th edition,2007), the content of which is hereby incorporated by reference.

Chemicals

12-NBD-stearate[12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazo) aminostearicacid] was from Avanti Polar Lipids (Alabaster, Ala.). BMS309403 was fromEMD Chemicals (San Diego, Calif.). Arachidonic acid was from CaymanChemical (Ann Arbor, Mich.). 48 virtually screened test compounds fromChemDiv, Inc. (Moscow, Russia). α-Truxillic acid 1-naphthyl ester(Compound 26) and γ-truxillic acid 1-naphthyl ester (Compound 49) weresynthesized at the Institute of Chemical Biology and Drug Discovery,Stony Brook University.

High Throughput Fluorescence Displacement Assay with NBD-Stearate

FABP5 was purified and delipidated as described previously (Kaczocha, M.et al. 2012). FABP5 (30 μg), NBD-stearate (1 μM), and a competitor testcompound were incubated in 30 mM Tris-HCl, 100 mM NaCl buffer (pH 7.6).Competitors included arachidonic acid, BMS309403, 48 test compounds fromChemDiv library, Compound 26 and Compound 49. The initial assay was runwith buffer (30 mM Tris-HCl buffer), negative controls (buffer andNBD-stearate), positive controls (buffer, NBD-stearate, FABP5), andexperimental wells with a variable test compound added (arachidonic acidor one of the 48 test compounds) at 10 μM. Test compounds that producedhigh inhibition and proved statistically significant were then added tothe fluorescent assay at 10 μM and tested in triplicate to verify theirsuccess. The most effective test compound and BMS309403 were measured inincreasing concentrations (0.01-50 μM), as were the Compound 26 andγ-truxillic acid 1-naphthyl ester, which were discovered following thetest. The fluorescent assays were tested in the wells of Microtest96-well Assay Plates, Optilux (BD Biosciences, Franklin Lakes, N.J.) andloss of fluorescence intensity was measured with a FLUOstar OPTIMAspectrofluorometer set to excitation and emission wavelengths of 460 nmand 544 nm, respectively. For the most effective test compounds, IC₅₀values were calculated with GraphPad Prism. GraphPad Prism was also usedto determine the K_(i) of these select competitors from the equationK_(i)═IC₅₀/(1+([NBD-stearate]/K_(d))). The K_(d) of NBD-stearate forFABP5 had been determined previously through incubating FABP5 withincreasing concentrations of NBD-stearate. One site binding analysis inGraphPad Prism indicated that the K_(d) of NBD-stearate for FABP5 was0.16 μM (Kaczocha, M. et al. 2012).

Patch-Clamp Electrophysiology in Brain Slices

Whole-cell-voltage clamp recordings of dorsal raphe (DR) serotonin(5-HT) neurons were performed as previously described [33]. Briefly, DRneurons were visualized using an upright microscope (BX 51 WI, Olympus,Tokyo, Japan) equipped with a differential interference contrast andinfrared imaging system. Somatic recordings from DR neurons wereobtained with path electrodes (3-5 mΩ) back-filled with potassiumgluconate based internal solution of the following composition: 120 mMpotassium gluconate, 10 mM KCl, 10 mM Na_(z)-phosphocreatine, 10 mMHEPES, 1 mM MgCl₂, 1 mM EGTA, 2 mM Na_(z)-ATP, 0.25 mM Na-GTP, pH 7.3(Adjusted with KOH; Osmolarity, 280 to 290 mOsmol/l). All the recordingswere conducted in the presence of GABA_(A) receptor antagonistpicrotoxin (100 μM). Excitatory postsynaptic currents (EPSCs) wereevoked with a single square stimulus (intensity, 1 to 10 V, duration,100 to 200 μs) delivered via a glass stimulating electrode. EPSCs wereamplified with a Multiclamp 700B (Molecular Devices, Union City, Calif.,USA) and acquired using pClamp 10 software (Molecular Devices).

Data Analysis

The amplitude of EPSCs was determined by measuring the average currentduring a 2-ms period at the peak of each EPSC and subtracted from thebaseline current determined during a 5-ms time window before thestimulus. All EPSC amplitudes were normalized to the mean baselineamplitude recorded for at least 10 min before drug application. Resultsin the text and figures are presented as mean±SEM. Statistical analysiswas conducted using the Student's paired t-test.

AEA Uptake

AEA uptake assays in wild-type and FABP5 knockdown HeLa cells wereperformed exactly as described (Kaczocha, M. et al. 2012).

FAAH Enzyme Assay

Enzyme assays measuring the hydrolysis of [¹⁴C]AEA in the presence ofCompound 26 or the FAAH inhibitor URB597 were carried out in HeLahomogenates expressing rat FAAH as described (Kaczocha, M. et al. 2009).

PPAR Transactivation

PPARα and PPARγ transactivation assays were performed in HeLa cellsexactly as described (Kaczocha, M. et al. 2012). Briefly, cells weretransfected with the PPAR reporter system, incubated with GW7647,rosiglitazone, or Compound 26 for 6 hrs, followed by measurement ofluciferase and β-galactosidase activity using a luminometer as described(Kaczocha, M. et al. 2012).

Animals

We used male C57Bl6 mice (22-30 g, Taconic Farms) for all experiments.The animals were group housed at room temperature and kept on a 12:12hour light:dark cycle with ad libitum access to water and food. Theanimals were habituated to the experimental room for one week beforetesting. All experiments were approved by the Stony Brook UniversityInstitutional Animal Care and Use Committee. The experimenter wasblinded to the treatment conditions of each animal.

Carrageenan-Induced Paw Edema and Thermal Hyperalgesia

Paw edema was induced by injecting 1% A-carrageenan (20 μl, in sterilesaline) into the plantar surface of the left hind paw and a controlsolution of saline into the right hind paw using a 27 gauge needle. Pawdiameters were measured before carrageenan injection and 4 hours afterinjection using digital electronic calipers (Fisher) and expressed tothe nearest ±0.01 mm. Compound 26 (20 mg/kg, i.p.) was dissolved inethanol:emulphor:saline (1:1:18), requiring sonication and gentleheating for solubilization, and administered 45 min prior to injectionof carrageenan. The cannabinoid receptor antagonists, rimonabant andSR144528 (3 mg/kg, i.p.), in ethanol:emulphor:saline (1:1:18), wereinjected 15 min before the FABP inhibitor. Edema is reported as thechange in paw diameter at 4 hr over the baseline. Changes in pawdiameter of saline-injected contralateral paws were negligible. Thermalhyperalgesia measured the latency to withdraw the paw from a focusedbeam of radiant heat applied to the plantar surface of the hind pawusing a Hargreaves plantar apparatus (Ugo Basile) set at an intensity of3.0. For each mouse, the average latencies consisted of three trialsspaced at least 5 minutes apart. The mice were habituated to the testchamber for 30 min. The cutoff time was set at 30 sec.

Formalin Test

Mice were habituated to the observation chamber (Plexiglas box, 25 cm×25cm×25 cm) for 30 min prior to formalin injection. The mice subsequentlyreceived an injection of formalin (2.5% in saline, 20 μl) into theplantar surface of the right hind paw using a 27 gauge needle. Theanimals were immediately placed back into the observation chamber andnocifensive behavior (time spent licking or biting the paw) was recordedfor 60 min. The formalin test consists of two phases with the firstphase (0-5 min) reflecting nociceptor activation and the second phase(15-45 min) reflecting an inflammatory pain response.

Statistical Analyses

Behavioral data are presented as means±S.E.M. for the vehicle andinhibitor-treated groups, each consisting of at least 6 animals.Statistical significance between vehicle and inhibitor groups wasdetermined using unpaired t-tests or one-way ANOVA followed by Dunnett'spost hoc analysis. In all cases, differences of p<0.05 were consideredsignificant.

High-Throughput Virtual Screening

A high-throughput virtual screening of over one million molecules fromthe ChemDiv subset of the ZINC database (http://zinc.docking.org) wasconducted on New York Blue, an 18 rack IBM Blue Gene/L massivelyparallel supercomputer located at Brookhaven National Laboratory usingDOCK version 6.5. Prior to docking, the most updated ChemDiv databasewas downloaded and presorted by rotatable bonds and split into 10subsets of ˜100,000 molecules using the DOCK database filter.Subsequently, an energy grid for FABP7 (PDB:1FE3) was generated usingthe grid program. Then, each molecule was flexibly docked to the FABP7grid (DOCK FLX protocol) and the single lowest-energy pose was retained.

Footprint-Based Rescoring

Following the high-throughput virtual screening, footprint-basedrescoring methodology developed by Rizzo and colleagues was implementedto enrich the library of docked molecules. First, the co-crystalizedligand oleic acid (reference) was minimized on the receptor Cartesiancoordinates within the binding pocket. This was implemented using both ahydrogen optimization followed by a weak restrained minimization(restraint of 10 kcal/mol). Following reference minimization, eachmolecule of the docked library was subsequently minimized in Cartesianspace using the restrained minimization protocol. Last, electrostatic,van der Waals, and hydrogen bond footprint similarity scores werecomputed using normalized Euclidian distance for each molecule dockedversus the reference using DOCK 6.5.

Database Clustering and Compound Selection

First, subsets 1 through 5 and subset 6 through 10 containing ˜500,000molecules were rank-ordered by the DOCK Cartesian energy (DCE) score.The top 45,000 of each combined subset of 500,000 molecules (˜10% total90,000 molecules) were then clustered using MACCS fingerprints, asimplemented in the program MOE with the tanimoto coefficient of 0.75.The resulting cluster heads obtained were then further rank-ordered by:(i) standard DOCK score (DCE_(VDW+ES)) (ii) van der Waals footprintsimilarity score (FPS_(VDW)), (iii) electrostatic footprint similarityscore (FPS_(ES)), (iv) H-bond footprint similarity score (FPS_(HB)), (v)the sum of van der Waals and electrostatic footprint similarity score(FPS_(VDW+ES)). The top 250 molecules rank-ordered by each criteria werethen plotted in MathPlotLib and examined by visual inspection andconsistency to the reference footprint. This method of analysis allowedus to both visually see key interactions within the binding pocket whilesimultaneously observing the magnitudes of those key interactions withinthe footprints for each molecule. Based on this approach, 48 compoundswere selected a purchased for biological testing against FABP5.Biological screening of these compounds for activity against FABP7 isunderway.

Redocking of FABP7 Hit Compounds into FABP5

The 48 molecules selected from the previous FABP7 virtual screening wereredocked using DOCK version 6.5 on a Quad Core Xeon Linux Server. Anenergy grid for FABP5 (PDB: 1B56, 2.05 {acute over (Å)}) was generatedusing the grid program. Then, each molecule was flexibly docked to theFABP5 grid (DOCK FLX protocol) and the single lowest-energy pose wasretained.

Example 1 In-Silico Lead Identification

The virtual screening (Irwin, J. J. et al. 2005) was conducted toelucidate new potential lead structures that could inhibit the transportof anandamide by FABP5 and FABP7. Utilizing a newly developed footprintsimilarity scoring (FPS) algorithm in DOCK 6.5 (Balius, T. E. et al.2011), a library of ligands was enriched based on their ability to matchkey interactions seen between the natural substrate oleic acid andFABP7. Recently, similar methodology has been employed for the discoveryof HIVgp41 inhibitors (Holden, P. M. et al. 2012).

High-throughput virtual screening in drug discovery has increasinglybecome a powerful and practical approach for pre-screening ligandlibraries for biologically relevant molecules (Kuntz, I.D. 1992;Jorgensen, W. L. 2004; Shoichet, B. K. 2004). Traditionally, dockingprograms attempt to approximate the intermolecular binding energybetween a ligand and a receptor. To save computational time, oftengrid-based approaches provide the best compromise between accuracy andsampling time (Meng, E. C. et al. 1992). Despite having moderate successrates, traditional docking typically favors larger molecules due todirect correlation between increasing van deer Walls energy and thenumber of atoms in a molecule (Balius, T. E. et al. 2011; Kuntz, I.D. etal. 1999). Often, small consideration of specific binding orientation isaccounted for; mostly translated through favorable electrostaticinteractions. Molecular footprints consist of two-dimensionalrepresentations of the ligand-receptor as a per-residue decomposition ofthe standard DOCK energy score (FIG. 2). Thus, we carried out ourvirtual screening based on the hypothesis that molecular footprintmatching between a docked library and a reference molecule wouldtranslate into a greater DOCK success rate, specifically the uniqueability to enrich for active compounds (positives) that areenergetically similar to a reference (Balius, T. E. et al. 2011).

The CB-1 receptor is predominately expressed in the brain and thus bothFABP5 and FABP7 were considered relevant targets for our virtualscreening. FABP5 or epidermal fatty acid binding protein (E-FABP) istypical dispersed throughout the body (tongue, adipose tissue, dendriticcell, mammary gland, brain neurons, kidney, liver, lung and testis) andfound abundantly in the epidermal cells of the skin. FABP7 or brainfatty acid binding protein (B-FABP) is typically expressed in highlevels during mid-term embryonic development but not present in neurons.Structural alignment (0.93 RMSD) of FABP7 (PDB: 1FE3, 2.8 Å) and FABP5(PDB: 1B56, 2.05 Å) revealed a 47% sequence identity and 66% similarity(FIG. 3). Furthermore, both FABP7 and FABP5 bind fatty acid substrateswith high affinity; although FABP7 typically shows higher bindingaffinity in-vitro. Thus, FABP7 was selected as our target for virtualscreening. Additional docking analysis has shown that DOCK scoresobtained from our FABP7 screening hits (48 compounds), tended to be morefavorable when re-docked into FABP5.

Footprint-Based Rescoring

High-throughput virtual screening utilizing the footprint rescoringmethod was conducted on FABP7 using oleic acid as the referencemolecule. This entailed: 1) grid setup and docking 2) minimization ofeach docked molecule and reference molecule on the receptor Cartesiancoordinates, 3) calculating the molecular footprints of all dockedmolecules and reference, 4) calculation of a footprint similarity score(FPS) for each of the docked molecules versus the reference oleic acid,5) MACCS fingerprint clustering, 6) rank-ordering based on each scoringcriteria, 7) analysis and selection of compounds from each of the 250cluster heads generated for each of the scoring criteria (FIG. 4) usingvisual inspection of binding poses and footprints.

First, subsets 1 through 5 and subset 6 through 10 containing ˜500,000molecules were rank-ordered by the DOCK Cartesian energy (DCE) score.The top 45,000 of each combined subset of 500,000 molecules (˜10% total90,000 molecules) were then clustered using MACCS fingerprints, asimplemented in the program MOE with the tanimoto coefficient of 0.75.The resulting cluster heads obtained were then further rank-ordered by:(i) standard DOCK score (DCE_(VDW+ES)), (ii) van der Waals footprintsimilarity score (FPS_(VDW)), (iii) electrostatic footprint similarityscore (FPS_(ES)), (iv) H-bond footprint similarity score (FPS_(HBOND)),(v) the sum or van der Waals and electrostatic footprint similarityscore (FPS_(VDW+ES)).

As a result of the virtual screening, 48 compounds were selected andassayed in-vitro against FABP5. Of the 48 compounds tested, 23 showed atleast 25% inhibition and 4 compounds, 19, 26, 27 and 31 (ChemDiv libraryID#: 5511-0235, 8009-2334, 8009-7646, and C075-0064), showedapproximately 50% inhibition or greater (FIG. 5). As seen in Table 1,Compound 26 had the highest correlation among all the leads to theelectrostatic footprint of oleic acid with favorable interaction atARG106, ARG126, TYR128 (FIG. 6). Furthermore, Compound 26 also had goodVDW overlap contributing to its low footprint score(FPS_(VDW+ES)=−56.76, −68.27) despite a significant clash seen in thecrystal structure for oleic acid at MET 115. Critical to the binding ofCompound 26, one free carboxylate similar to that observed in oleic acidwas essential, specifically for the binding to ARG106, ARG126, TYR128sequence.

TABLE 1 Dock energy scores for FABP7 and FABP5 and experimental valuesfor FABP5 % I K_(i) ID Method DCE_(FABP7) FP_(VDW+ES) FP_(VDW) FP_(ES)DCE_(FABP5) CDiv ID (μM) 19 FPS_(VDW+ES) −56.56 1.09 0.81 0.28 −64.3867.5  ND 5511-0235 26 FPS_(VDW+ES) −56.76 1.01 0.87 0.14 −68.27 78.121.19 N/A 27 FPS_(ES) −53.34 1.21 1.00 0.21 −65.91 49.57 ND 8009-7646 31FPS_(VDW+ES) −53.77 1.12 0.73 0.39 −71.20 49.93 ND C075-0064 Avg. −55.111.11 0.85 0.26 −67.44 61.28 —

Example 2 Synthesis of α-Truxillic Acid Monoesters

As shown below (Scheme 1), starting from E-(trans)-cinnamic acidirradiation in the solid state promotes the [2+2] cycloaddition toafford the head to tail dimer α-truxillic acid in good yields andexcellent purity. Further modification of the α-truxillic acid to yieldα-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono esters proceedsthrough the di-acyl chloride intermediate followed by addition of analcohol or amine substituent to form the ester and amide productsrespectively. Mono and di substitution products are seen forsubstitution with 1-napthol at a ratio of 2.5 in favor of the monosubstitution. Workup with 1N HCl converts the remaining acyl chloride tothe corresponding free carboxylic acid.

α-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid (α-truxillic acid)

E-cinnamic acid, 1.272 g (8.59 mmol) was placed into a pyrex dish andexposed to light at 350 nM and an intensity of 280 mW/cm² for 1 weekwith periodic shaking. This process was performed in the solid state andcould be monitored by ¹H NMR. After completion the white solid waswashed with diethylether (200 mL) and allowed to dry. The solid was thenrecrystallized from pure ethanol to give alpha-truxillic acid 1.006 g(3.39 mmol), 79% yield as a white solid. mp 276-277° C. (lit. 274-278°C.)¹H NMR (300 MHz, DMSO-d₆): δ 12.12 (s, 2H), 7.32 (m, 8H), 7.24 (m,2H), 4.28 (dd, J=7.2 Hz, 10.1 Hz, 2H) 3.81 (dd, J=7.2 Hz, 10.1 Hz, 2H).¹³C NMR (75 MHz, DMSO-d₆): δ 173.00, 139.47, 128.19, 127.67, 126.69,46.17, 41.06. Data is consistent with literature values (Yang, J. L. etal. 2007)

α-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-(1-napthol) ester(Compound 26)

To 500 mg (1.69 mmol) of pure alpha-truxillic acid, 20 mL oftetrahydrofuran was added along with 1.01 mL (11.81 mmol) of oxalylchloride. While stirring 1 drop of DMF was added and the solution wasallowed to stir at room temperature for 1 hour. After completion, thereaction was concentrated in-vacuo to remove all volatiles. To the crudeyellow solid made previously, 20 mL of tetrahydrofuran was addedfollowed by the addition of 0.19 mL (1.34 mmol) of triethylamine and 84mg (0.69 mmol) of DMAP. While stirring 173 mg (1.20 mmol) of 1-naptholpreviously dissolved in 10 mL of tetrahydrofuran was added dropwise. Thereaction was allowed to stir overnight at room temperature and quenchedwith 1 N HCl upon completion. The reaction mixture was then extractedusing 100 mL of dichlorormethane and washed with 100 mL of brine. Theresulting organic layer was dried with MgSO₄ and concentrated in-vacuoto give a brownish oil which was then subsequently purified using flashchromatography with 20% ethyl acetate in hexanes followed by 30% ethylacetate hexanes to give the α-2,4-diphenyl-cyclobutane-1,3-dicarboxylicacid mono-(1-napthol) ester 210 mg (0.50 mmol), 42% yield as a whitesolid. mp 195° C. ¹H NMR (500 MHz, DMSO-d₆): δ 12.24 (s, 1H), 7.88 (d,J=8.5 Hz, 1H), 7.73 (d, J=8.5 Hz, 1H), 7.57 (d, J=7.0 Hz, 2H), 7.52 (d,J=7.5 Hz, 2H), 7.48-7.37 (m, 6H), 7.34-7.27 (m, 3H), 7.06 (d, J=8.0 Hz,1H), 6.38 (d, J=7.5 Hz, 1H) 4.61-4.49 (m, 3H), 3.98 (dd, J=7.0 Hz J=10.0Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 172.73, 170.93, 146.17, 139.23,139.03, 133.92, 128.74, 128.25, 128.19, 127.89, 127.67, 127.40, 126.90,126.47, 126.22, 125.85, 125.45, 121.13, 117.86, 46.17, 41.54, 41.11.HRMS (ESI) m/e calculated for C₂₈H₂₃O₄H⁺:423.1589. Found: 423.1596(Δ=1.7 ppm).

Example 3 Synthesis of γ-Truxillic Acid Monoesters

As shown below (Scheme 2), starting from α-truxillic acid isomerizationat high temperature and formation of the anhydride by addition of aceticanhydride provides the glutaric anhydride γ-truxillic acid precursor.This anhydride intermediate can then afford anyγ-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono esters or monoamides by activation with DMAP at room temperature and addition of analkyl or aromatic alcohol or amine.

3-Oxabicyclo[3.1.1]heptane-2,4-dione, 6,7-diphenyl (γ-TruxillicAnhydride)

To 600 mg (2.02 mmol) of pure α-truxillic acid, 775 mg of anhydroussodium acetate and 5 mL of acetic anhydride was added to a 20 mL roundbottom flask. The solution was refluxed at 150° C. for 24 hours. Aftercompletion the solution was allowed to cool to room temperature. Thewhite solid was washed and filtered with 100 mL of water. The resultingsolid was dried and then first dissolved in chloroform (10 mL) and thenupon addition of 100 mL of ethanol white crystals formed. The whitecrystals were dried and yielded 398 mg (1.43 mmol), 71% of the desiredcompound. mp 187° C. (lit. 190° C.)¹H NMR (300 MHz, CDCl₃): δ 7.51-7.39(m, 4H), 7.37-26 (m, 4H), 7.09-7.06 (m, 2H), 4.34 (t, J=5.7 Hz, 1H) 4.07(d, J=5.1 Hz, 2H), 3.99 (s, 1H).

γ-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-(1-napthyl) ester(Compound 49)

To 50 mg (0.18 mmol) of pure 3-Oxabicyclo[3.1.1]heptane-2,4-dione,6,7-diphenyl, 1 mL of tetrahydrofuran was added along with 29 mg (0.20mmol) of 1-napthol. While stirring 0.03 mL (0.20 mmol) ofdiisopropylethylamine was added and the solution was allowed to stir atroom temperature for 15 hours. After completion, the reaction wasconcentrated in-vacuo to remove all volatiles. The reaction mixture wasthen extracted using 100 mL of dichlorormethane and washed with 100 mLof brine. The resulting organic layer was dried with MgSO₄ andconcentrated in-vacuo to give a brownish solid which was thensubsequently purified using flash chromatography with 20% ethyl acetatein hexanes followed by 30% ethyl acetate hexanes to give theγ-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-(1-napthol) ester61 mg (0.14 mmol), 80% yield as a white solid. ¹H NMR (500 MHz,DMSO-d₆): δ 12.27 (b, 1H), 7.87 (d, J=8.5 Hz, 1H), 7.73 (d, J=8.0 Hz,1H), 7.48-7.24 (m, 13H), 6.88 (d, J=8.0 Hz, 1H), 6.46 (d, J=7.5 Hz, 1H),4.59 (t, J=10.5 Hz, 1H), 4.47 (t, J=10.0 Hz, 1H), 4.37 (t, J=10.5 Hz,1H), 3.85 (t, J=10.5 Hz, 1H).

γ-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-(2-napthyl) ester(Compound 50)

To 50 mg (0.18 mmol) of pure 3-Oxabicyclo[3.1.1]heptane-2,4-dione,6,7-diphenyl, 1 mL of tetrahydrofuran was added along with 29 mg (0.20mmol) of 2-napthol. While stirring 0.03 mL (0.20 mmol) ofdiisopropylethylamine was added and the solution was allowed to stir atroom temperature for 15 hours. After completion, the reaction wasconcentrated in-vacuo to remove all volatiles. The reaction mixture wasthen extracted using 100 mL of dichlorormethane and washed with 100 mLof brine. The resulting organic layer was dried with MgSO₄ andconcentrated in-vacuo to give a brownish solid which was thensubsequently purified using flash chromatography with 20% ethyl acetatein hexanes followed by 30% ethyl acetate hexanes to give theγ-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-(2-napthyl) ester70 mg (0.17 mmol), 92% yield as a white solid. ¹H NMR (300 MHz,DMSO-d₆): δ 7.85 (d, J=7.2 Hz, 1H), 7.76 (d, J=9.0 Hz, 1H), 7.66 (d,J=7.2 Hz, 1H), 7.51-7.24 (m, 12H), 6.77 (s, 1H), 6.43 (d, J=8.7 Hz, 1H),4.55 (t, J=10.8 Hz, 1H), 4.40 (t, J=10.0 Hz, 1H), 4.16 (t, J=10.5 Hz,1H), 3.84 (t, J=10.2 Hz, 1H).

Example 4 Compound 26 Bound to FABP7

The free carboxylate of Compound 1 binds primarily to TYR129 and ARG126similarly to fatty acid substrates (FIG. 7). Furthermore, ARG106 hassome interaction as seen in the electrostatic (ES) foot print. However,the long distance between the carboxylate and ARG106 lowers thisinteraction and suggests possible water mediated hydrogen bonding.Lastly, MET115 provides steric hindrance with the natural substrateoleic acid (Ki 0.35 μM±0.04 FABP7) and provides selectivity for thetruxillic acid core.

Example 5 Determination of K_(i)

Determination of Ki was derived from calculation using the K_(d) ofstearic acid (Scheme 1). BMS309403 which has been assayed previouslyutilizing this assay was determined to have a K_(i) of 0.89 μM±0.31against FABP5. In comparison, both theα-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-(1-napthol) ester(Compound 26) and γ-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acidmono-(1-napthol) ester (Compound 49), each isolated as a mixture ofenantiomers, were tested in triplicate and yielded inhibitory dosedependent K_(i) values of 0.93 μM±0.07 and 0.75 μM±0.07 against FABP5,respectively (FIGS. 8A and 8B). Thus theγ-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-(1-napthol) esterwas the better inhibitor. The inhibitory dose dependent K_(i) values ofBMS309403 was 0.75 μM±0.164 against FABP5 (FIG. 8C).

Example 6 Effects of Compound 26 on Glutamate-Mediated SynapticTransmission

Activation of cannabinoid CB1 receptors inhibits glutamatergic synaptictransmission in numerous brain areas, including the dorsal root ganglionDR (Haj-Dahmane, S et al. 2009). Therefore, to test whether Compound 26exhibits agonist properties at CB1 receptors, we examined its effects onthe amplitude of glutamate-mediated excitatory postsynaptic currents(EPSCs) recorded from DR 5-HT neurons. We found that bath application ofCompound 26 (10 μM) did not inhibit the amplitude of EPSCs (107±6.7% ofbaseline, n=8 (FIG. 8A-B). Such a finding suggests that Compound 26 isnot a CB1 receptor agonist.

Example 7 Effect of Compound 26 Upon AEA Uptake in Cells

We have previously shown that FABPs are intracellular carriers thatshuttle endocannabinoids and related N-acylethanolamines tointracellular sites, such as FAAH for hydrolysis (Kaczocha, M. et al.2209; Kaczocha, M. et al. 2012). Pharmacological or genetic inhibitionof FABPs reduces AEA catabolism in cells, confirming an essential rolefor these proteins in endocannabinoid inactivation. Therefore, weexamined whether the novel FABP inhibitor Compound 26 reducesFABP-mediated AEA uptake in cells. Indeed, Compound 26 significantlyinhibited cellular AEA accumulation. Confirming its selectivity forFABPs, Compound 26 failed to reduce AEA uptake in cells bearing aknockdown of FABP5 (FIG. 9A), the main FABP expressed in HeLa cells.Additionally, Compound 26 does not inhibit FAAH (FIG. 9B). Collectively,these results indicate that Compound 26 is a selective FABP inhibitor.

Example 8 Compound 26 Produces Antinociceptive and Anti-InflammatoryEffects in Mice

Similar to cannabinoid receptor agonists, inhibitors of endocannabinoidinactivation produce anti-inflammatory and antinociceptive effects(Cravatt, B. F. et al. 2001; Lichtman, A. H. 1990). Importantly, FAAHinhibitors lack the untoward psychotropic effects of cannabinoidreceptor agonists (Cravatt, B. F. et al. 2004), highlighting thetherapeutic advantage of pharmacologically targeting endocannabinoidinactivation. Because inhibition of AEA transport to FAAH reduces AEAinactivation, we hypothesized that FABP inhibitors may likewise possessantinociceptive and anti-inflammatory properties. Therefore, we examinedthe effects of Compound 26 using two nociceptive models: the formalintest and carrageenan-induced thermal hyperalgesia. In the formalin test,injection of formalin results in the induction of two temporallydistinct phases of pain with the first phase (0-5 min) representingnociceptor activation and the second phase (15-45 min) representinginflammatory pain and central sensitization. As shown in FIG. 10A,Compound 26 significantly reduced nocifensive behavior only during thefirst phase of the formalin test.

We next explored whether Compound 26 alleviates inflammatory paininduced by intraplantar injection of A-carrageenan. Indeed, Compound (20mg/kg, i.p.) significantly reduced carrageenan-induced thermalhyperalgesia (FIG. 10B) and paw edema (FIG. 10C). To establish acannabinoid receptor-mediated mechanism of action, mice were pretreatedwith a combination of the cannabinoid receptor 1 and 2 antagonists,rimonabant and SR144528, respectively. The antinociceptive andanti-edematous effects of Compound 26 were completely reversed byrimonabant and SR144528 (FIGS. 10B and 10C). Previous reports indicatethat α-truxillic acid derivatives activate peroxisomeproliferator-activated receptor γ (PPARγ), which alongside PPARα,modulate nociception (Steri, R. et al. 2010; Loverme, J, et al. 2006;Churi, S. B. et al. 2008). In our hands, Compound 26 served as a weakagonist at both receptors, displaying ˜2-fold activation of PPARα and˜3-fold activation of PPARγ (FIG. 11).

Example 10 Additional Derivatives

An additional aspect of the invention provides analogues of Compounds 1,2, and 3 that are active as FABP inhibitors. The analogs of Compounds 1,2, and 3 described below in Schemes 4-10 have analogous activity toCompound 26.

The [2+2] cycloaddition of the derivatives of trans-cinammic acid areknown to proceed through light induced photocyclization. Thus thecorresponding substituted phenyl analogues of α-truxillic acid can besynthesized and alternatively converted to γ-truxillic analogues byisomerization. R_(n) can include any alkyl, aryl, heteroaryl, hydroxyl,thiol, amine, amide, carbamate, urea, halogen, hydrazide, etc. (Scheme4).

Both α and γ truxillic acids are symmetrical molecules and thus areinherently enantiopure by symmetry. Upon functionalization however,desymmetrization creates chirality induced by the equal chance fornucleophllic acyl substitution to occur at either carbonyl. Thus by theuse an enantiopure nucleophile, two distereomers can be formed asopposed to two enantiomers facilitating separation of the correspondingdistereomers by either flash chromatography or preparative HPLC.(S)-(−)-1-phenylethanol can serve both as a good chiral auxiliary andprotection group with selective removal by Pd/C.

For γ-truxillic acids, specifically the glutaric anhydride intermediate,addition of (S)-(−)-1-phenylethanol can be accomplished at roomtemperature by addition of DMAP (Scheme 5).

For α-truxillic acids, addition of (S)-(−)-1-phenylethanol to producethe mono-ester is accomplished by first forming the di-acyl chlorideintermediate with oxalyl chloride and then subsequent nucleophilic acylsubstitution in the presence of TEA (Scheme 6).

Any of compounds 51A, 51B, 52A or 52B, once separated from theircorresponding diastereomer, are used as intermediates en route to avariety of enantiopure analogs that have analogous activity to Compound26. Compounds 51A, 51B, 52A or 52B may be reacted with oxalyl chloridein the presence of naphthalen-1-ol to form both enantiomers, separable,of Compound 26 or Compound 49.

For example, upon the separation of enantiopure truxillic acid 51A,further modification is performed by first generating the acyl chloridefollowed by the addition of an amine or alcohol with TEA to afford thehetero di-substituted compound. Selective deprotection of the benzylicgroup by Pd/C thereby affords enantiopure mono esters or amides (Scheme7).

Further diversification of the truxillic acid leads is investigated byselective reduction of the free carboxylic acid with borane dimethylsulfide which affords the corresponding primary alcohol of, for example,51A (Scheme 8). To the alcohol, commercial available acyl chlorides isadded in the presence of TEA followed by Pd/C deprotection to afford“reversed ester” analogues. In addition, any aryl alcohol is coupled viathe Mitsunobu reaction to form the corresponding ether analogues afterdeprotection by Pd/C.

Substitution of the free carboxylic acid by an alpha hydroxy or alphaketo acid provides additional water mediated hydrogen bonding. Thus,these structures are promising as a divergence from the traditionaltruxillic acid core. Following selective etherification, the freecarboxylic acid is selectively reduced to the corresponding alcohol.After reduction, oxidation with PCC affords the aldehyde which can thenbe subjected to the Strecker reaction followed by hydrolysis to form thecorresponding alpha hydroxy acids (Scheme 9). Further modification tothe alpha keto acid is achieved by Pummerer oxidation before the lasthydrolysis step (Scheme 9).

Additional analogs are synthesized according to the protocols in Scheme10. Enantiopure compound 51 is reduced with borane dimethyl sulfidewhich affords the corresponding primary alcohol. Treatment with sodiumazide results in the alkyl azide, which is reduced to the primary amine.The primary amine is converted to a variety of analogs (Scheme 10).

Additional analogs are synthesized according to the protocols in Scheme11. The enantiopure aldehyde (synthesized by methods shown in Scheme 9)is converted to a variety of analogs (Scheme 11).

Example 11 Additional Route to Enantiopure Analogs

One way of resolving enantiomers is to convert them into theircorresponding diastereomers and separate them by known purificationtechniques (as shown in Schemes 5 and 6). An additional method is toconvert the two enantiomers of, for example, Compound 26 into thecorresponding glucamine salts (Scheme 12), which are separable based ontheir difference solubility. Crystallization and neutralization allowsfor the isolated of either pure enantiomer (Harrington, P. J. et al1997).

Example 12 Compounds 53 and 54

Compounds 53 and 54 were synthesized according to the synthetic methodsdescribed hereinabove and have analogous FABP inhibition activity toCompound 26.

3-(naphthalen-1-ylcarbamoyl)-2,4-diphenylcyclobutanecarboxylic acid(Compound 53)

¹H NMR (300 MHz, DMSO-d₆) δ 12.12 (b, 1H), 9.87 (s, 1H), 7.33 (d, J=7.8Hz, 1H), 7.66 (d, J=8.1 Hz, 1H), 7.53 (d, J=7.2 Hz, 2H), 7.27-7.47 (m,12H), 7.08 (d, J=7.2 Hz, 1H), 4.64 (dd, J=2.7 Hz, J=7.5, 1H), 4.32 (dd,J=3.0 Hz J=7.5 Hz, 1H), 3.59, (t, J=6.3, 1H), 1.74 (m, 1H).

3-((naphthalen-2-yloxy)carbonyl)-2,4-diphenylcyclobutanecarboxylic acid(Compound 54)

¹H NMR (300 MHz, DMSO-d₆) δ 2.14 (b, 1H), 7.86 (d, J=8.1 Hz, 1H), 7.77(d, J=8.7 Hz, 1H), 7.67 (d, J=7.2 Hz, 1H), 7.27-7.54 (m, 10H), 7.23 (d,J=6.6 Hz, 2H), 6.77 (s, 1H), 6.48 (d, J=9.0 Hz, 1H), 4.56 (t, J=10.5 Hz,1H), 4.47 (t, J=10.8 Hz, 1H), 4.28 (t, J=10.2 Hz, 1H), 3.98 (t, J=10.2Hz, 1H); MS/MS (ESI− negative mode) m/e calculated for C₂H₂₃O₄—H:421.1439. Found: 421.1.

Example 13 Additional In Vivo Mouse Studies and Pharmacokinetic Data

Compounds 26 and 54 (20 mg/kg, i.p.) reduced carrageenan-induced thermalhyperalgesia and paw edema in mice (FIG. 16A). Compounds 26 and 54 alsoreduced the first and second phases of formalin-induced nociception inmice (FIG. 16B). Compound 26 reduced acetic acid-induced writhing inmice (FIGS. 16C-D). Compound 26 also elevated brain levels of theendocannabinoid anandamide (AEA) (FIG. 17). Compound 26 was administeredby a single injection and plasma and brain concentrations were analyzedover 24 hours.

Discussion Virtual Screening and Initial Testing

High-throughput virtual screening in drug discovery has increasinglybecome a powerful and practical approach for pre-screening ligandlibraries for biologically relevant molecules. Traditionally, dockingprograms attempt to approximate the intermolecular binding energybetween a ligand and a receptor. To save computational time, oftengrid-based approaches provide the best compromise between accuracy andsampling time. Despite having moderate success rates, traditionaldocking typically favors larger molecules due to direct correlationbetween increasing van deer Walls energy and the number of atoms in amolecule. Often, only a small consideration of specific bindingorientation is accounted for, mostly translated through favorableelectrostatic interactions. A method of rescoring compounds based ontheir molecular footprints has been implemented into the program DOCK6.5. Molecular footprints are two-dimensional representation of theligand-receptor as a per-residue decomposition of the standard DOCKenergy score. Thus, a virtual screening was carried out based on thehypothesis that molecular footprint matching between a docked libraryand a reference molecule would translate into a greater DOCK successrate based on the unique ability to enrich for true positives.

Before starting the virtual screening process, consideration was takento select the most relevant biological target. The CB-1 receptor ispredominately expressed in the brain and thus both FABP5 and FABP7 wereconsidered relevant targets. FABP5 or epidermal fatty acid bindingprotein (E-FABP) is typical dispersed throughout the body (tongue,adipose tissue, dendritic cell, mammary gland, brain neurons, kidney,liver, lung and testis) and found abundantly in the epidermal cells ofthe skin. FABP7 or brain fatty acid binding protein (B-FABP) istypically expressed in high levels during mid-term embryonic developmentbut not present in neurons. A structural comparison reveals that FABP7(PDB: 1FE3, 2.8 Å) and FABP5 (PDB: 1B56, 2.05 Å) share 45% sequenceidentity and 63% similarity.

Furthermore, both FABP7 and FABP5 bind fatty acid substrates with highaffinity: although FABP7 typically shows higher binding affinityin-vitro. Interestingly, cross docking shows that DCE scores for FABP5tend to be on average lower than those obtained for FABP7. Thus, FABP7was selected as our target for virtual screening. High-throughputvirtual screening utilizing the footprint rescoring method was conductedon FABP7 using oleic acid as the reference molecule. This entailed: 1)grid setup and docking, 2) minimization of each docked molecule andreference molecule on the receptor Cartesian coordinates, 3) calculatingthe molecular footprints of all docked molecules and reference, 4)calculation of a footprint similarity score (FPS) for each of the dockedmolecules versus the reference oleic acid, 5) MACCS fingerprintclustering, 6) rank-ordering based on each scoring criteria, 7) analysisand selection of compounds from each of the 250 cluster heads generatedfor each of the scoring criteria. As a result of the virtual screening,48 compounds were selected by scoring criteria for in-vitro assay.

A sample footprint analysis of the reference compound oleic acid and atest compound called ZINC00695558 from the ChemDiv library. Footprintsimilarity scoring involved van der Waal, Coulombic, and hydrogenbonding forces. The methods employed for Footprint (FPS) scoringeliminated approximately 1,057,000 compounds with tile identification of48 compounds selected for binding assay.

The binding assay utilized an established fluorescence displacementassay. The degree to which the test compounds displaced NBD-stearate (1μM) from FABPs is shown in FIG. 12. The first two samples, the bufferand NBD-stearate do not give appreciable fluorescence while theNBD-stearate plus purified FABP5 gives an appreciable fluorescencesignal. The fourth sample is the positive control where arachidonic acid(1 μM), a fatty acid that binds strongly to FABP5 (K_(i) 0.13 μM)decreases the signal. Each sample in this screen was measured induplicate and approximately ⅓ of the test compounds appeared to causedisplacement of NBD-stearate with a concomitant decrease influorescence. Four of the most potent (Compounds 19, 26, 27, and 31)were selected for further evaluation (FIGS. 16 and 17) and statisticalanalysis.

The best four FABP5 inhibitors discerned by the initial screen wherethen rerun in the NBD-fluorescent assay with replicate measurements at10 μM. Inhibition of NBD-stearate binding to FABP5 by these compoundswas highly significant with the napthol truxillic derivative the mostpotent (Compound 26). In control experiments it was observed that thefour test compounds (19, 26, 27, or 31) did not fluoresce under theassay conditions at 10 μM, nor did 10 μM concentrations of thesecompounds quench the fluorescence of 16 μM NBD-stearate, that is 16times the concentration used in the routine assay (FIG. 13). The mostpotent inhibitor (compound 26) was synthesized to confirm the structureof the sample provided from ChemDiv. This α-napthol truxillic acidderivative was tested over a wide concentration range and a K_(i) of0.93±0.08 μM (FIG. 8A) while γ-napthol triuxillic derivative (Compound49) was more potent with a K_(i) of 0.75±0.07 (FIG. 8B), howeverCompound 49 was less soluble than Compound 26. The BMS compound had aK_(i) of 0.75±0.164 μM (FIG. 8C).

The two most potent compounds, 26 and 49 (the α- and γ-truxillic acid1-naphthyl esters), discerned from our in silico and biologicalscreening, belong to a class of compounds that have been found to haveanti-inflammatory and anti-nociceptive (Chi, Y. et al. 2005; Chi, Y. etal. 2006). Heretofore, the mechanism by which these effects weremediated was unknown. However, we can speculate that these compoundsinhibit the transport of anandamide and other fatty acid ethanolamides,such as palmitoylethanolamide and oleoylethanolamide. These increasedNAE levels would lead to greater signaling at the cannabinoid andpotentiate NAE-mediated hypoalgesic and anti-inflammatory effects,indicating that modulation of NAE signaling may represent a therapeuticavenue for the treatment of pain.

Truxillic Acid Analogues

The design of truxillic acid based compounds targeting early stage(neurogenic pain response) and late stage (inflammatory pain response)has coincidently been studied recently based on the structure of thenatural product (−)-incarvillateine. This natural product was firstisolated from the plant species Incarvillea sinensis, which has beenknown in traditional Chinese medicine to treat rheumatism and pain.Interestingly, the isolated (−)-incarvillateine was found to possessantinociceptive properties on the same level as morphine.

Recently research (Chi, Y. et al. 2005) was carried out to identifypotential leads for commercialization purposes focusing on di-esters anddi-carboxylic acid derivatives. Despite the lack of targetidentification, α and β-truxillic acid di-ester and di-carboxylic acidderivatives were designed, synthesized and tested against early and latestage pain in formalin induced mouse models to study the effects of eachanalogue in-vivo. The results of the SAR study clearly showed thatα-truxillic acid alone provided the best antinociceptive agent in bothearly and late stage pain. Although the para-hydroxyl functionalizationon the phenyl rings slightly improved late stage pain relief itdrastically reduced early stage pain relief.

As described herein, both α-2,4-diphenyl-cyclobutane-1,3-dicarboxylicacid mono-esters and γ-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acidmono-esters are reversible inhibitors of FABPs. These compounds bind toFABPs and block the shuttling of endocannabinoids within the cell andthereby increase the endogenous levels of the endocannabinoid anandamideby circumventing degradation by FAAH. Increased levels of anandamideresult in the activation of the CB1 pathway leading to antinociceptivepain relief and reduction of inflammation which has been shown in aformalin induced mouse model (in-vivo results))

To further our library of truxillic acid based FABP inhibitors, otherknown FABP5 inhibitors was studied. BMS480404 is reported to have a Kiof 33 nM±2 nM against FABP5 and a K_(i) of 2.5 nM±0.1 nM against FABP4determined by a 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS)displacement assay (FIG. 14) (McDonnell, P. et al. 2006). Note that theassays are different and thus the K_(i) determined may not be comparableto that obtained with the NBD-stearate assay. Interestingly, thecarboxylic acid derivative of BMS480404 is shown to possess an improvedKi of 3 nM±1 nM and the alpha keto acid produced a K_(i) of less than 2nM. Thus incorporation of the alpha keto acid functionality, as inBMS480404-5, into the truxillic acid core may provide addition bindingthrough water mediated hydrogen bonding to ARG 106 (FIG. 15). Theinteraction of the carboxylate of Compound 1 with ARG106 can be seenbelow with an electrostatic energy between −2 to −3 kcal/mol (FIG. 6).In addition, the compound BMS480404 contains two ethers and thus seemeda good alternative to the ester which could potentially be cleaved byesterases. Lastly, chirality may play an important role in binding andthus synthesis of enatiopure ligands may provide useful, as can alreadybe seen by differences in K_(i) for the α and γ2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-esters,respectively.

As described herein, α-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acidmono-(1-napthol) ester (Compound 1) andγ-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-(1-napthol) ester(Compound 2) were selected based on a virtual screening. These leadsprovided excellent activity in-vitro against FABP5 with dose dependentKi values of 0.93 μM±0.08 and 0.75 μM±0.07 respectively. In addition,α-2,4-diphenyl-cyclobutane-1,3-dicarboxylic acid mono-(1-napthol) ester(Compound 2) provided excellent results in formalin induced mice models.Building upon these results, other α and γ truxillic acid basedanalogues targeting FABP's have analogous activity to Compound 2.

SUMMARY

The current study identifies novel small molecule inhibitors of FABP5and FABP7 by virtual screening employing DOCK and FPS. Despite havingdissimilar distributions within the body, the structural similaritybetween FABP5 and FABP7 was shown to be as high as 66% with key bindingresidues fully conserved. We therefore choose to use FABP7 to identifypotential lead compounds using DOCK and FPS, and performed similaritymatching between the VDW and ES footprints of oleic acid our referencesubstrate, and over one million docked small molecules. Forty-eightmolecules were identified in the virtual screen and subsequently assayedagainst FABP5 using a high-throughput fluorescent displacement assay.Overall, four compounds were identified as potential competitiveinhibitors of FABP5. The most potent compound, Compound 26, was found topossess a diphenyl-cyclobutane core characteristic of the known naturalproduct (−)-incarvillateine.

A novel α-truxillic acid 1-naphthyl mono-ester, Compound 26, wassynthesized and the FABP5 NBD-stearate displacement assay of thiscompound showed a sub-micro molar Ki value. The resynthesized γ-form oftruxillic acid 1-naphthyl mono-ester (Compound 49) also showed sub-micromolar efficacy against FABP5, which was considerably more potent thanCompound 26, probably due to the difference in purity. Compound (α-form)and Compound 49 (γ-form) were found to be as potent as BMS309403, a wellknown FABP inhibitor. Thus with both our virtual and biologicalscreening, truxillic acid mono-esters were identified as a unique classof compounds that target FABPs.

Following biological screening and binding analyses of these inhibitors,we have shown that the novel FABP inhibitor Compound 26 producesantinociceptive and anti-inflammatory effects in mice. These findingsare in agreement with a previous study demonstrating that someα-truxillic acid derivatives exhibited antinociceptive properties,although the mechanism of action was not identified (Chi, T. M. et a.2006). It was subsequently reported that certain derivatives ofα-truxillic acid activate PPARγ (Steri, R. et al. 2010). Although ourwork demonstrates that Compound 26 behaves as a weak agonist at PPARαand PPARγ, its antinociceptive effects were abolished by cannabinoidreceptor antagonists. Therefore, the antinociceptive effects of Compound26 likely resulted from potentiation of endocannabinoid signaling ratherthan activation of PPAR receptors. Taken together, our results establishFABPs as novel targets for antinociceptive drug development. In additionto the FABP transporters described here, heat shock protein 70, albumin,and a truncated fatty acid amide hydrolase protein have also beenreported as intracellular shuttles for AEA (Fu, J. et al. 2011;Maccarrone, M. et al. 2010) and this area has been recently review(Fowler, C. J. 2012). These studies show the potential for the design ofeven more potent inhibitors that will be selective for individual FABPs.

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1. The compound having the structure:

wherein R₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O)C(═O)OR₁₃ or —C═C—R₁₃, wherein R₁₃ and R₁₄ areeach, independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or combine toform a cycloalkyl or heterocyclyl; R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁and R₁₂ are each independently, H, halogen, —NO₂, —CN, —NHR₁₅, NR₁₅R₁₆,—SR₁₅, —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NR₁₅R₁₆, -alkyl-OR₁₅, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀, alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloheteroalkyl, aryl, heteroaryl,or heterocyclyl; when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆,R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ isother than —C(═O)OR₁₃ where R₁₃ is —CH₃, —CH₂CH₃, tolyl or propyl1-bromo-1-methylpropanoyloxybutyl ester, or —C(═O)NR₁₃R₁₄ where one ofR₁₃ or R₁₄ is phenyl and the other is —H, or both of R₁₃ and R₁₄ are —H;and when one of R₁ or R₂ is —C(═O)OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈, R₉,R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other than—C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the other is(2-methylmercapto)phenyl; or an enantiomer or pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1 having thestructure:

wherein R₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O)C(═O)OR₁₃ or —C═C—R₁₃, wherein R₁₃ and R₁₄ areeach, independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or combine toform a cycloalkyl or heterocyclyl; R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁and R₁₂ are each independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆,—SR₁₅, —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NR₁₅R₁₆, -alykl-OR₁₅, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloheteroalkyl, aryl, heteroaryl,or heterocyclyl; when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆,R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ isother than —C(═O)OR₁₃ where R₁₃ is —CH₃, —CH₂CH₃, —(CH₂)₄CH₃,—(CH₂)₇CH₃, —CH₂(CH₃)₂, —CH₂C(O)CH₃, tolyl, 1-Naphthol or propyl1-bromo-1-methylpropanoyloxybutyl ester, or —C(═O)NR₁₃R₁₄ where one ofR₁₃ or R₁₄ is phenyl and the other is —H, or both of R₁₃ and R₁₄ are —H;and when one of R₁ or R₂ is —C(═O)OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈, R₉,R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other than—C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the other is(2-methylmercapto)phenyl; or an enantiomer or pharmaceuticallyacceptable salt thereof.
 3. The compound of claim 1 having thestructure:

wherein R₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O) OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O)C(═O)OR₁₃ or —C═C—R₁₃, wherein R₁₃ and R₁₄ areeach, independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or combine toform a cycloalkyl or heterocyclyl; R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁and R₁₂ are each independently, H, halogen, —NO₂, —CN, —NR₁₅R₁₆, —SR₁₅,—SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆,-alykl-OR₁₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,heteroaryl, or heterocyclyl; wherein R₁₅ and R₁₆ are each,independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl; whenone of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ andR₁₂ are each H, then the other of R₁ or R₂ is other than —C(═O)OR₁₃where R₁₃ is —CH₂CH₃ or propyl 1-bromo-1-methylpropanoyloxybutyl ester;when one of R₁ or R₂ is —C(═O)OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀,R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other than—C(═O)NR₁₃R₁₄, where one of R₁₃ and R₁₄ is —H and the other is(2-methylmercapto)phenyl; or an enantiomer or pharmaceuticallyacceptable salt thereof.
 4. The compound of claim 1 having thestructure:

wherein R₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)NR₁₃R₁₄, -alkyl-OC(═O)OR₁₃,-alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)NR₁₃R₁₄,-alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄, —C(—OH)C(═O)OR₁₃,—C(═O)C(═O)OR₁₃ or —C═C—R₁₃, wherein R₁₃ and R₁₄ are each,independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or combine toform a cycloalkyl or heterocyclyl; R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁and R₁₂ are each independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆,—SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆,-alykl-OR₁₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,heteroaryl, or heterocyclyl; wherein R₁₅ and R₁₆ are each,independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl; whenone of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ andR₁₂ are each H, then the other of R₁ or R₂ is other than —C(═O)OR₁₃where R₁₃ is —CH₃ or tolyl or —C(═O)NR₁₃R₁₄ where both of R₁₃ and R₁₄are —H; or an enantiomer or pharmaceutically acceptable salt thereof. 5.The compound of claim 4, wherein one of R₁ or R₂ is —C(═O)R₁₃,—C(═O)OR₁₃, —C(═O)NHR₁₃, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃,-alkyl-C(═O)NHR₁₃, -alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, or-alkyl-NHR₁₃, wherein R₁₃ is aryl or heteroaryl; and the other of R₁ orR₂ is —C(═O)OR₁₃, wherein R₁₃ is H.
 6. The compound of claim 5, whereinone of R₁ or R₂ is

and the other of R₁ or R₂ is —C(═O)OH.
 7. (canceled)
 8. (canceled) 9.The compound of or claim 4, wherein one of R₁ or R₂ is -alkyl-C(═O)R₁₃,-alkyl-C(═O)OR₁₃, -alkyl-C(═O)NHR₁₃, -alkyl-OC(═O)OR₁₃,-alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NHR₁₃, wherein R₁₃ is H, aryl orheteroaryl; and the other of R₁ or R₂ is —C(—OH)C(═O)OR₁₃, or—C(═O)C(═O)OR₁₃, wherein R₁₃ is H or alkyl.
 10. The compound of claim 9,wherein one of R₁ or R₂ is

and the other of R₁ or R₂ is


11. (canceled)
 12. The compound of claim 1 having the structure:

or a pharmaceutically acceptable salt thereof.
 13. The compound of claim1 having the structure:

or a pharmaceutically acceptable salt thereof.
 14. A process forproducing the compound of claim 4 comprising: (a) contacting a compoundhaving the structure:

with acetic anhydride in the presence of sodium acetate so as to producea compound having the structure:

(b) reacting the product of step (a) with a nucleophile (Nuc) in a firstsuitable solvent in the presence of an amine base so as to produce amixture of enantiomers having the structures:


15. The method of claim 14, wherein the nucleophile used in step (b) is

Or the nucleophile used in step (b) is a chiral nucleophile; or thenucleophile used in step (b) is (S)-(−)-1-phenylethanol. 16-18.(canceled)
 19. The method of claim 15, wherein the products of step (b)are


20. The method of claim 19, further comprising (c) separating thediastereomeric products of step (b) to produce enantiopure compoundshaving the structure:

(d) reacting a product of step (c) with a coupling reagent in thepresence of a nucleophile in a second suitable solvent so as to produceenantiopure compounds having the structure:

(e) reacting the product of step (d) with hydrogen in the presence ofpalladium on carbon to produce an enantiopure compound having thestructure:


21. (canceled)
 22. A method of inhibiting the activity of a Fatty AcidBinding Protein (FABP) comprising contacting the FABP with a compoundhaving the structure:

wherein R₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O) OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH) C(═O) OR₁₃, —C(═O)C(═O)OR₁₃ or —C═C—R₁₃, wherein R₁₃ and R₁₄ areeach, independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or combine toform a cycloalkyl or heterocyclyl; R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁and R₁₂ are each independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆,—SR₁₅, —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆,-alykl-OR₁₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,heteroaryl, or heterocyclyl; wherein R₁₅ and R₁₆ are each,independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, aryl, heteroaryl, or heterocyclyl; when one of R₁ or R₂ is—C(═O)OH or —C(═O)OCH₃, then the other of R₁ or R₂ is other than—C(═O)OR₁₃ where R₁₃ is alkyl, heteroalkyl, substituted phenyl orbenzyl, —C(═O)NHR₁₃R₁₄ where one of R₁₃ or R₁₄ is —H, phenyl orsubstituted phenyl and the other is —H, or —C(═O)NR₁₃R₁₄ where R₁₃ andR₁₄ combine to form a piperidine or morpholine; or a pharmaceuticallyacceptable salt thereof.
 23. The compound of claims 22, wherein when oneof R₁ or R₂ is —C(═O)OH or —C(═O)OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈, R₉,R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other than—C(═O)OR₁₃ where R₁₃ is alkyl, heteroalkyl, substituted phenyl orbenzyl, —C(═O)NHR₁₃R₁₄ where one of R₁₃ or R₁₄ is —H, phenyl orsubstituted phenyl and the other is —H, or —C(═O)NR₁₃R₁₄ where R₁₃ andR₁₄ combine to form a piperidine or morpholine.
 24. The method of claim22, wherein the compound has the structure:

wherein R₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(—O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH) C(═O) OR₁₃, —C(═O)C(═O)OR₁₃ or —C═C—R₁₃, wherein R₁₃ and R₁₄ areeach, independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or combine toform a cycloalkyl or heterocyclyl; R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁and R₁₂ are each independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆,—SR₁₅, —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alykl-NR₁₅R₁₆, -alkyl-OR₁₅, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;wherein R₁₅ and R₁₆ are each, independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, heteroalkyl, cycloheteroalkyl, aryl, heteroaryl,or heterocyclyl; when one of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆,R₇, R₈, R₉, R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ isother than —C(═O)OR₁₃ where R₁₃ is —CH₃, —CH₂CH₃, —(CH₂)₄CH₃,—(CH₂)₇CH₃, —CH₂(CH₃)₂, —CH₂C(O)CH₃, tolyl, 1-Naphthol or propyl1-bromo-1-methylpropanoyloxybutyl ester, or —C(═O)NR₁₃R₁₄ where one ofR₁₃ or R₁₄ is phenyl and the other is —H, or both of R₁₃ and R₁₄ are —H;and when one of R₁ or R₂ is —C(═O)OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈, R₉,R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other than—C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the other is(2-methylmercapto)phenyl; or an enantiomer or pharmaceuticallyacceptable salt thereof.
 25. The method of claim 22, wherein thecompound has the structure:

wherein R₁ and R₂ are different and are each —C(═O)R₁₃, —C(═O)OR₁₃,—C(═O)NR₁₃R₁₄, -alkyl-C(═O)R₁₃, -alkyl-C(═O)OR₁₃, -alkyl-C(═O)NR₁₃R₁₄,-alkyl-OC(═O)OR₁₃, -alkyl-OC(═O)R₁₃, -alkyl-OR₁₃, -alkyl-NR₁₃R₁₄,-alkyl-NHC(═O)R₁₃, -alkyl-NHC(═O)OR₁₃, -alkyl-NHC(═O)R₁₃,-alkyl-NHC(═O)NR₁₃R₁₄, -alkyl-NHC(═S)NR₁₃R₁₄, -alkyl-NHC(═NR₁₃)NR₁₃R₁₄,—C(—OH)C(═O)OR₁₃, —C(═O)C(═O)OR₁₃ or —C═C—R₁₃, wherein R₁₃ and R₁₄ areeach, independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or combine toform a cycloalkyl or heterocyclyl; R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁and R₁₂ are each independently, H, halogen, —NO₂, —CN, —NHR₁₅, —NR₁₅R₁₆,—SR₁₅, —SO₂R₁₅, —OR₁₅, —CO₂R₁₅, CF₃, -alkyl-NHR₁₅, -alkyl-NHR₁₅R₁₆,-alykl-OR₁₅, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,heteroaryl, or heterocyclyl; wherein R₁₅ and R₁₆ are each,independently, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroalkyl, cycloheteroalkyl, aryl, heteroaryl, or heterocyclyl; whenone of R₁ or R₂ is —C(═O)OH and R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ andR₁₂ are each H, then the other of R₁ or R₂ is other than —C(═O)OR₁₃where R₁₃ is —CH₃, —CH₂CH₃, tolyl or propyl1-bromo-1-methylpropanoyloxybutyl ester, or —C(═O)NR₁₃R₁₄ where one ofR₁₃ or R₁₄ is phenyl and the other is —H, or both of R₁₃ and RD are —H;and when one of R₁ or R₂ is —C(═O)OCH₃ and R₃, R₄, R₅, R₆, R₇, R₈, R₉,R₁₀, R₁₁ and R₁₂ are each H, then the other of R₁ or R₂ is other than—C(═O)NR₁₃R₁₄ where one of R₁₃ and R₁₄ is —H and the other is(2-methylmercapto)phenyl; or an enantiomer or pharmaceuticallyacceptable salt thereof.
 26. The method of claim 22, wherein thecompound inhibits binding of an FABP ligand to the FABP; or wherein theFABP ligand is an endocannabinoid ligand; or wherein the FABP ligand isanandamide (AEA) or 2-arachidonoylglycerol (2-AG).
 27. (canceled) 28.(canceled)
 29. A method of identifying an agent that inhibits theactivity of a Fatty Acid Binding Protein (FABP) comprising contacting aFatty Acid Binding Protein (FABP) expressed in the CNS with the agentand separately with a compound having the structure

and comparing the FABP inhibitory activity of the agent with the FABPinhibitory activity of the compound to identify an agent where FABPinhibitory activity is greater than that of the compound.